Vectors for the diagnosis and treatment of solid tumors including melanoma

ABSTRACT

The present invention is directed to the isolation and use of super-infective, tumor-specific vectors that are strains of parasites including, but not limited to bacteria, fungi and protists. In certain embodiments the parasites include, but are not limited to, the bacterium Salmonella spp., such as  Salmonella typhimurium , the bacterium  Mycobacterium avium  and the protozoan  Leishmania amazonensis . In other embodiments, the present invention is concerned with the isolation of super-infective, tumor-specific, suicide gene-containing strains of parasites for use in treatment of solid tumors.

This application is a continuation application of U.S. patentapplication Ser. No. 08/658,034 filed Jun. 4, 1996, which issued on Feb.20, 2001 as U.S. Pat. No. 6,190,657, and which in turn is acontinuation-in-part application of U.S. patent application Ser. No.08/486,422 filed Jun. 7, 1995, now abandoned, which is incorporated byreference herein in its entirety.

1. FIELD OF THE INVENTION

The present invention is concerned with the isolation and use ofsuper-infective, tumor-specific, attenuated strains of parasitesincluding, but not limited to, bacteria, fungi and protists. In certainembodiments the parasites include the bacterium Salmonella spp., such asSalmonella typhimurium, the bacterium Mycobacterium avium, and theprotozoan Leishmania amazonensis, for the diagnosis and treatment ofsarcomas, carcinomas, and other solid tumor cancers. In otherembodiments, the present invention is concerned with the isolation anduse of super-infective, tumor-specific, suicide gene-containing strainsof parasites.

2. BACKGROUND OF THE INVENTION

Citation or identification of any reference in Section 2 of thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

A major problem in the chemotherapy of solid tumor cancers is thedelivery of therapeutic agents, such as drugs, in sufficientconcentrations to eradicate tumor cells while at the same timeminimizing damage to normal cells. Thus, studies in many laboratoriesare directed toward the design of biological delivery systems, such asantibodies, cytokines, and viruses for targeted delivery of drugs,pro-drug converting enzymes, and/or genes into tumor cells. Houghton andColt, 1993, New Perspectives in Cancer Diagnosis and Management 1:65-70; de Palazzo,et al., 1992a, Cell. Immunol. 142:338-347; de Palazzoet al., 1992b, Cancer Res. 52: 5713-5719; Weiner, et al., 1993a, J.Immunotherapy 13:110-116; Weiner et al., 1993b, J. Immunol.151:2877-2886; Adams et al., 1993, Cancer Res. 53:4026-4034; Fanger etal., 1990, FASEB J. 4:2846-2849; Fanger et al., 1991, Immunol. Today12:51-54; Segal, et al., 1991, Ann N.Y. Acad. Sci. 636:288-294; Segal etal., 1992, Immunobiology 185:390-402; Wunderlich et al., 1992; Intl. J.Clin. Lab. Res. 22:17-20; George et al., 1994, J. Immunol.152:1802-1811; Huston et al., 1993, Intl. Rev. Immunol. 10:195-217;Stafford et al., 1993, Cancer Res. 53:4026-4034; Haber et al., 1992,Ann. N.Y. Acad. Sci. 667:365-381; Haber, 1992, Ann. N.Y. Acad. Sci. 667:365-381; Feloner and Rhodes, 1991, Nature 349:351-352; Sarver and Rossi,1993, AIDS Research & Human Retroviruses 9:483-487; Levine andFriedmann, 1993, Am. J. Dis. Child 147:1167-1176; Friedmann, 1993, Mol.Genetic Med. 3:1-32; Gilboa and Smith, 1994, Trends in Genetics10:139-144; Saito et al., 1994, Cancer Res. 54:3516-3520; Li et al.,1994, Blood 83:3403-3408; Vieweg et al., 1994, Cancer Res. 54:1760-1765;Lin et al., 1994, Science 265:666-669; Lu et al., 1994, Human GeneTherapy 5:203-208; Gansbacher et al., 1992, Blood 80:2817-2825; Gastl etal., 1992, Cancer Res. 52:6229-6236.

Because of their biospecificity, such systems could in theory delivertherapeutic agents to tumors. However, it has become apparent thatnumerous barriers exist in the delivery of therapeutic agents to solidtumors that may compromise the effectiveness of antibodies, cytokines,and viruses as delivery systems. Jain, 1994, Scientific American 7:58-65(Jain). For example, in order for chemotherapeutic agents to eradicatemetastatic tumor cells, they must

a) travel to the tumors via the vasculature;

b) extravasate from the small blood vessels supplying the tumor;

c) traverse through the tumor matrix to reach those tumor cells distalto the blood supply; and

d) interact effectively with the target tumor cells (adherence,invasion, pro-drug activation, etc).

Although antibodies and viruses can express specific recognition sitesfor tumor cells, they are dependent solely upon the forces of diffusionand convection in order to reach these sites. According to Jain:

An agent that destroys cancers cells in a culture dish should, intheory, be able to kill such cells in the body. . . . Sadly, however,the existing pharmacopoeia has not markedly reduced the number of deathscaused by the most common solid tumors in adults, among them cancers ofthe lung, breast, colon, rectum, prostate, and brain. . . . Before ablood-borne drug can begin to attack malignant cells in a tumor, it mustaccomplish three critical tasks. It has to make its way into amicroscopic blood vessel lying near malignant cells in the tumor, exitfrom the vessel into the surrounding matrix (the interstitium), andfinally, migrate through the matrix to the cells. Unfortunately, tumorsoften develop in ways that hinder each of these steps.

Jain points out that blood vessels supplying tumors are irregular andconvoluted in shape so that blood flow is frequently restricted comparedto that in normally vascularized tissue. In addition, there is anunusually high interstitial pressure in many tumors that counteracts theblood flow. Jain further points out that the two chief forces governingthe transport of agents to tumor cells via the circulatory system areconvection (the transport of molecules by a stream of flowing fluid),and diffusion (the movement of molecules from an area of highconcentration to an area of low concentration). Since tumors are oftennon-uniformly vascularized, many cells in the tumors receive nutrientsthrough the process of diffusion through the matrix. Jain and coworkersobtained data suggesting that “a continuously supplied monoclonalantibody having a molecular weight of 150,000 daltons could take severalmonths to reach a uniform concentration in a tumor that measured onecentimeter in radius and had no blood supply in its center.”

2.1. Bacterial Infections and Cancer

Regarding bacteria and cancer, an historical review reveals a number ofclinical observations in which cancers were reported to regress inpatients with bacterial infections. Nauts et al., 1953, Acta Medica.Scandinavica 145:1-102, (Suppl. 276) state:

The treatment of cancer by injections of bacterial products is based onthe fact that for over two hundred years neoplasms have been observed toregress following acute infections, principally streptococcal. If thesecases were not too far advanced and the infections were of sufficientseverity or duration, the tumors completely disappeared and the patientsremained free from recurrence.

Shear, 1950, J. A.M.A. 142:383-390 (Shear), observed that 75% of thespontaneous remissions in untreated leukemia in the Children's Hospitalin Boston occurred following an acute episode of bacterial infection.Shear stated:

Are pathogenic and non-pathogenic organisms one of Nature's controls ofmicroscopic foci of malignant disease, and in making progress in thecontrol of infectious diseases, are we removing one of Nature's controlsof cancer?

Subsequent evidence from a number of research laboratories indicatedthat at least some of the anti-cancer effects are mediated throughstimulation of the host immune system, resulting in enhancedimmuno-rejection of the cancer cells. For example, release of thelipopolysaccharide (LPS) endotoxin by Gram negative bacteria such asSalmonella triggers release of tumor necrosis factor, TNF, by cells ofthe host immune system, such as macrophages, Christ et al., 1995,Science 268:80-83. Elevated TNF levels in turn initiate a cascade ofcytokine-mediated reactions which culminate in the death of tumor cells.In this regard, Carswell et al., 1975, Proc. Natl. Acad. Sci. USA72:3666-3669, demonstrated that mice injected with bacillusCalmette-Guerin (BCG) have increased serum levels of TNF and thatTNF-positive serum caused necrosis of the sarcoma Meth A and othertransplanted tumors in mice. Further, Klimpel et al., 1990, J. Immunol.145:711-717, showed that fibroblasts infected in vitro with Shigella orSalmonella had increased susceptibility to TNF.

As a result of such observations as described above, immunization ofcancer patients with BCG injections is currently utilized in some cancertherapy protocols. See Sosnowski, 1994, Compr. Ther. 20:695-701; Barthand Morton, 1995, Cancer 75 (Suppl. 2) :726-734; Friberg, 1993, Med.Oncol. Tumor. Pharmacother. 10:31-36 for reviews of BCG therapy.

2.2. Parasites and Cancer Cells

Although the natural biospecificity and evolutionary adaptability ofparasites has been recognized for some time and the use of theirspecialized systems as models for new therapeutic procedures has beensuggested, there are few reports of, or proposals for, the actual use ofparasites as vectors.

In this regard, Pidherney et al., 1993, Cancer Letters 72:91-98(Pidherney et al.) and Alizadeh et al., 1994, Infect. Immun.62:1298-1303 (Alizadeh et al.) have provided evidence that thepathogenic free-living amoeba, Acanthamoeba castellani, has tumorcidalcapabilities toward human tumor cells, including melanoma, when added totumor cells growing in culture or when injected directly into tumors innude mice. Pidherney et al. conclude:

The feasibility of utilizing the tumorcidal properties ofpathogenic/free-living amoebae and their cell-free products in thetreatment of drug-resistant or radio-resistant tumors warrants furtherinvestigation.

However, Pidherney et al. also point out that such pathogenic/freeliving amoebae can exist either as free-living organisms feeding onbacteria or as opportunistic pathogens producing life-threateningmeningoencephalitis or blinding keratitis.

Thus, it is readily apparent that for any parasite to be effective as atherapeutic vector, for example, for human tumors, the benefit of theparasite as a vector must outweigh its risk as a pathogen to thepatient. Therefore, although Pidherney et al. and Alizadeh et al.demonstrated cytotoxicity of pathogenic amoebae toward tumor cells, andfurther suggested their use in the treatment of drug-resistant andradio-resistant tumors, they offered no solution for the inherentpathogenicity of these organisms once injected into cancer patients.Furthermore, they offered no method, e.g., genetic selection forisolating super-infective, tumor-specific strains of pathogenic amoebaenor did they suggest insertion into the amoebael genome of geneticconstructs containing inducible genes for the synthesis and secretion ofpro-drug converting enzymes and/or suicide gene products.

Likewise, Lee et al., 1992, Proc. Natl. Acad. Sci. USA 89:1847-1851 (Leeet al.) and Jones et al., 1992, Infect. Immun. 60:2475-2480 (Jones etal.) isolated mutants of Salmonella typhimurium that were able to invadeHEp-2 (human epidermoid carcinoma) cells in vitro in significantlygreater numbers than the wild type strain. The “hyperinvasive” mutantswere isolated under conditions of aerobic growth of the bacteria thatnormally repress the ability of wild type strains to invade HEp-2 animalcells. However, Lee et al. and Jones et al. did not suggest the use ofsuch mutants as therapeutic vectors, nor did they suggest the isolationof tumor-specific bacteria by selecting for mutants that show infectionpreference for melanoma or other cancers over normal cells of the body.Without tumor-specificity or other forms of attenuation, suchhyperinvasive Salmonella typhimurium as described by Lee et al. andJones et al. would likely be pan-invasive, causing wide-spread infectionin the cancer patient. Further, without selection for tumor specificityor employment of other forms of attenuation, use of such bacteria astherapeutic vectors would increase the risk of pan-infection and septicshock to the cancer patient.

Pan et al., 1995, Nature Medicine 1:471-477 (Pan et al.) described theuse of Listeria monocytogenes as a vaccine for the immunization of miceagainst lethal challenges with tumor cells expressing the same antigenexpressed by the Listeria vaccine. In addition, they showed regressionof established tumors when immunized after tumor development in anantigen specific T-cell-dependent manner. However, Pan et al. did notshow that Listeria monocytogenes could be used as a tumor specificvector, which would target and amplify within the tumor. Rather, Pan etal. showed that recombinant Listeria monocytogenes has the ability todeliver a foreign antigen to the immune system and to involvecell-mediated immunity against the same antigen.

Sizemore et al., 1995, Science 270:299-302 (Sizemore et al.) describedthe use of attenuated Shigella bacteria as a DNA delivery vehicle forDNA-mediated immunization. Sizemore et al. showed that an attenuatedstrain of Shigella invaded mammalian cells in culture and delivered DNAplasmids containing foreign genes to the cytoplasm of the cells. Foreignprotein was produced in the mammalian cells as a result of theprocedure. The Shigella vector was designed to deliver DNA to colonicmucosa, providing a potential oral and mucosal DNA immunizationprocedure as well as other gene immunotherapy strategies. However,Sizemore et al. did not suggest the use of such attenuated Shigella astumor vectors in that they could be used to target tumors and therebyexpress genes within them. Rather, Sizemore et al. envisioned its use invaccination therapy following oral delivery and invasion of the mucosa.

Clostridium was previously investigated as a potential therapeuticvector for solid tumors. The propensity of spores of the obligateanaerobe Clostridium to germinate in necrotic tissues is well known.Tetanus and gas gangrene result from successful colonization of necrotictissue by pathogenic members of this genus.

Parker et al., 1947, Proc. Soc. Exp. Biol. Med. pp. 461-467 first showedthat direct injection of spores of Clostridium histolyticus into atransplantable sarcoma growing in a mouse caused oncolysis, i.e.,liquification, as well as regression of the tumor. In general theprocess of Clostridium-mediated oncolysis was accompanied by acutetoxicity and death of the mice. Malmgren and Flanigan, 1955, Cancer Res.15:473 demonstrated that mice bearing mammary carcinomas, hepatomas, andother tumors died within 48 hrs of intravenous injection of Clostridiumtetani spores, whereas control, non-tumor bearing animals wereasymptomatic for 40 days. Möse and Möse, 1964, Cancer Res. 24:212-216(Möse and Möse) described the colonization and oncolysis of tumors byClostridium butyricum, strain M-55, a non-pathogenic soil isolate. Möseand Möse established the lack of human pathogenicity of the M-55 strainby administering spores to themselves, as reported by Carey et al.,1967, Eur. J. Cancer 3:37-46. Using Clostridium butyricum strain M-55,Möse and Möse reported that intravenous injections of spores causedoncolysis of the mouse Erlich ascites tumor, growing experimentally as asolid tumor. Aerobic spore-forming organisms—e.g., Bacillusmesentericus, Bacillus subtilis, which were prepared in a similarmanner, did not show any oncolysis under the same conditions. Möse andMöse concluded that the clostridial oncolysis was restricted toanaerobic areas of the tumors because of the anaerobic metabolicrequirements of the bacteria.

Gericke and Engelbart, 1964, Cancer Res. 24:217-221 showed thatintravenously injected spores of strain M-55 produced extensive lysis ofa number of different tumors, but with shortened survival times of theClostridium-treated, tumor-bearing animals compared to non-treatedtumor-bearing animals. Further, they found that “metastases in organs orlymph nodes were unaffected by the spores unless the metastatic tumorshad reached a considerable size.”

Thiele et al., 1964, Cancer Res. 24:222-233 showed that intravenouslyinjected spores of a number of species of nonpathogenic Clostridia,including M-55, localized and germinated in tumor tissue, but not innormal tissues of the mouse. Thiel et al., 1964, Cancer Res. 24:234-238found that spore treatment produced no effect when administered early inthe development of the tumor, i.e., when the tumors were of small size.While the spores caused oncolysis in tumors of sufficient size, therewas no effect in smaller tumors or metastases. The animals regularlydied during oncolysis. Carey et al., 1967, Eur. J. Cancer 3:37-46,concluded that small tumors and metastases had been noted to beresistant to oncolysis whereas large neoplasms were particularlyfavorable. Thus, the qualitative differences in germination of sporeswere likely to be not a characteristic of neoplastic and normal tissuesper se, but related to physiologic and biochemical conditions foundwithin large tumor masses.

Recent molecular genetic studies have focused on anaerobic bacteria ofthe genus Clostridium as potential tumor vectors. Fox et al., 1996, GeneTherapy 3:173-178 using a Clostridium expression vector were able totransform the E. coli cytosine deaminase gene into Clostridiumbeijerincki, which resulted in increased cytosine deaminase activity inthe growth medium supernatant and cell extracts of transformedclostridial bacteria. Such supernatants, when added to cultures of mouseEMT6 carcinoma made the cells sensitive to 5-fluorocytosine, presumablythrough its conversion to the toxic 5-fluorouracil. Similarly, Minton etal., 1996, FEMS Microbiol. Rev. 17:357-364 inserted the E. colinitroreductase gene into Clostridium beijerincki and were able to detectexpression of the gene in an in vivo murine tumor model through the useof antibodies directed against the E. coli nitroreductase gene. Thenitroreductase gene product activates CB1954, a potent alkylating agent.

Nothing in any of the above references (or any other references known tothe present inventors) suggests the use of any microorganisms, otherthan the obligate anaerobe Clostridium, as a potential therapeuticvector for solid tumors.

2.3. Attenuated Salmonella Spp.

Bacon et al., 1950, Br. J. Exp. Path. 31:703-713; Br. J. Exp. Path.31:714-724; 1951, Br. J. Exp. Path. 32:85-96 demonstrated thatattenuation of Salmonella for virulence in mice can be achieved throughauxotrophic mutations, i.e., through the use of mutants which lack theability to synthesize precursor molecules necessary for growth. Morespecifically, the authors showed that purine-requiring (Pur⁻) auxotrophsof Salmonella were attenuated in mice.

Hoiseth and Stocker, 1981, Nature 291: 238-239 showed that Salmonellatyphimurium auxotrophic mutants with requirements for aromatic aminoacids (Aro⁻) were attenuated for virulence in C57BL mice. Further, Su etal., 1992, Microbiol. Pathogenesis 13:465-476 showed that one such Aro⁻mutant, the attenuated antigen carrier strain of Salmonella typhimurium,SL3261, was useful as a vaccine. The Shiga toxin B-subunit/hemolysin A(C-terminus) fusion protein was expressed and underwent extracellularexport resulting in antigen-specific immune responses in mice inoculatedwith these bacteria.

O'Callaghan et al., 1988, Infect. Immun. 56:419-423 characterizedSalmonella typhimurium that were both Aro- and Pur- and found thatalthough they were highly attenuated in BALB/c mice, they persisted forseveral weeks in the livers and spleens following i.v. injections. Theywere found to be ineffective as vaccines when administered either orallyor i.v.

Johnson et al., 1991, Mol. Microbiol. 5:401-407 (Johnson et al.)demonstrated that attenuation in Salmonella virulence can be achievedthrough mutations in the heat shock inducible protein HtrA, a serineprotease. Chabalgoity et al., 1996, Mol. Microbiol. 19:791-801,demonstrated that such attenuated htrA- Salmonella typhimurium wereuseful as live vaccines.

However, none of the references by Bacon et al., Hoiseth and Stocker,O'Callaghan et al., Johnson et al., Su et al. 1992, Chabalgoity et al.1996, nor any of the studies referred to in Table 4, infra, suggest thatsuch avirulent strains of Salmonella typhimurium would survive andproliferate within solid tumors, nor that such avirulent mutants mightbe used as vectors for solid tumor therapy.

2.4. Objectives of the Invention

The problems associated with the many physical barriers for delivery oftherapeutic agents to solid tumors provide clear and difficult obstaclesin the design of effective delivery systems. Thus, there has been a longfelt need in the art to provide delivery systems which are able toovercome these obstacles.

It is an object of the present invention to use and to provide moreadvanced biological vectors such as parasites having several distinctadvantages as a novel delivery system, some of which are listed below,as well as to meet the challenges of tumor therapy.

Antibiotic Sensitivity: It is an advantage for a tumor-specificparasitic vector to be sensitive to exogenously administeredantibiotics. Parasites, such as bacteria, can be eradicated within theirhosts by the administration of antibiotics. Such antibiotic sensitivityallows for the eradication of the parasite from the cancer patient'sbody upon completion of the therapeutic protocol.

Biospecificity: It is an advantage for a vector to express specificityfor its target cell, e.g., a tumor cell. The more specificity, of thevector for the tumor cell, the lower the inoculum necessary foreffective therapy, thereby reducing the risk of septic shock orpan-infection to the cancer patient. Parasites show a great degree ofnatural biospecificity, having evolved to utilize a variety of specificrecognition and invasion mechanisms. (For general discussions onbiospecificity see: Falkow, 1991, Cell 65:1099-1102; Tumomanen, 1993,Am. Soc. Microbiol. 59:292-296).

Mutant Isolation and Genetic Manipulation: It is an advantage, in thedesign and isolation of a parasite as a tumor-specific, therapeuticvector, for the parasite to be amenable to genetic manipulation.Parasites with haploid genomes and short generation times, for example,bacteria such as Salmonella typhimurium and enteroinvasive Escherichiacoli, can be readily subjected to mutagenesis followed by enrichmentprocedures for the isolation of strains with desired new characteristics(see generally, Neidhardt et al., (ed.) 1987, Escherichia coli andSalmonella typhimurium, Cellular and Molecular Biology. American Societyfor Microbiology, pp 990-1033. Furthermore, the methods for the geneticanalysis and stable introduction of genetic constructs into thesebacteria are well known to the science of molecular genetics.

Chemotaxis: A chemotactic response toward cancer cells is an advantagefor a tumor-specific vector, for example as a stimulus for the vector toinvade through a basement membrane matrix such as that produced byendothelial cells in the vasculature, or as a stimulus for the vector toseek out cancer cells surrounded by tumor matrix. Chemotactic responsesin parasites and commensalists or mutualists, particularly in bacteriasuch as Escherichia coli and Salmonella typhimurium, are welldocumented. For a review of chemotaxis see Macnab, 1992, Ann. Rev.Genet. 26:131-158.

Replication Within Target Cells: The ability to replicate within targetcells is an advantage for a tumor-specific vector. Such an abilityallows for amplification of the therapeutic vector number within theinfected cancer cell, thus increasing the therapeutic effectiveness ofthe vector. Progeny of vectors within cancer cells further infectsurrounding or distant cancer cells, thus amplifying the vector numberwithin the tumor cell population.

Anaerobic and Aerobic Metabolism: The ability to express invasive andamplification capacities under either aerobic or anaerobic conditions isan advantage for a tumor-specific vector. Solid tumors generally containvascularized, oxygen-rich areas as well as necrotic oxygen-poor areas. Avector that is functional in both such environments would be able toreach a larger portion of tumor cells than one that can function in onlyone environment, such as, for example, an obligate anaerobe or aerobe.

3. SUMMARY OF THE INVENTION

The present invention provides compositions and methods for delivery ofgenes and/or gene products to and/or into target mammalian cells invitro or in vivo. The genes and/or gene products are delivered bymicroorganism vectors, including bacteria, fungal and protozoanparasites, which are selected and/or genetically engineered to bespecific to a particular type of target mammalian cell. In a preferredembodiment, the vectors function under both aerobic and anaerobicconditions, are super-infective, tumor-specific microorganisms usefulfor diagnosis or treatment of sarcomas, carcinomas, lymphomas or othersolid tumor cancers, such as germ line tumors and tumors of the centralnervous system, including, but not limited to, breast cancer, prostatecancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer,testicular cancer, thyroid cancer, astrocytoma, glioma, pancreaticcancer, stomach cancer, liver cancer, colon cancer, and melanoma.

Vectors useful for the methods of the present invention include but arenot limited to Borrelia burgdorferi, Brucella melitensis, Escherichiacoli, enteroinvasive Escherichia coli, Legionella pneumophila,Salmonella typhi, Salmonella typhimurium, Shigella spp., Streptococcusspp., Treponema pallidum, Yersinia enterocohtica, Chlamydia trachomatis,Listeria monocytogenies, Mycobacterium avium, Mycobacterium bovis,Mycobacterium tuberculosis, BCG, Mycoplasma hominis, Rickettsiaequintana, Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystiscarnii, Eimeria acervulina, Neospora caninum, Plasmodium falciparum,Sarcocystis suihominis, Toxoplasma gondii, Leishmania amazonensis,Leishmania major, Leishmania mexacana, Leptomonas karyophilus,Phytomonas spp., Trypanasoma cruzi, Encephahtozoon cuniculi, Nosemahelminthorum, Unikaryon legeri.

As used herein, Salmonella typhimurium encompasses all Salmonellaspecies. It has long been recognized that the various “species” of thegenus Salmonella are in fact a single species by all acceptable criteriaof bacterial taxonomy. The single species is now designated “Salmonellaenterica”. F. Neidhardt (ed.), Escherichia coli and Salmonella, 1996,Volume I, pp. xx, ASM Press, Wash. D.C.

An embodiment of the present invention is to provide methods for theisolation of super-infective, attenuated, tumor-specific mutants ofmicroorganisms such as bacterial, fungal and protozoan parasites.Further, the present invention provides methods for use of thesemicroorganisms in the diagnosis and treatment of malignant and/ormetastatic solid tumor cancers, such as melanoma or colon cancer.Moreover, these mutant parasites may express specific gene products,some of which are secreted into the cytoplasm or vacuolar space of theinfected cell.

The present invention provides methods for the isolation ofsuper-infective target cell-specific microorganisms. In particularembodiments, the invention provides for the isolation and use ofsuper-infective, tumor-specific strains of parasites such as thebacterium Salmonella spp., including S. typhimurium, the bacteriumMycobacterium avium, and the protozoan Leishmania amazonensis. Thetumor-specific vectors can also contain suicide genes.

One embodiment of the present invention provides methods for theisolation of and compositions comprising super-infective, tumor-specificmutants of Salmonella spp., e.g., Salmonella typhimurium, and for theiruse in the diagnosis and treatment of sarcomas, carcinomas, melanomas,colon cancer, and other solid tumor cancers. Another embodiment of thepresent invention provides methods for the isolation of and compositionscomprising super-infective, tumor-specific mutants of Salmonella spp.containing a suicide gene. In a specific embodiment, the suicide gene isthymidine kinase from Herpes simplex virus or cytosine deaminase fromEscherichia coli or human microsomal p450 oxidoreductase.

Another embodiment of the present invention provides methods for theisolation of and compositions comprising super-infective, tumor-specificmutants of the protozoan, Leishmania amazonensis and for their use inthe diagnosis and treatment of sarcomas, carcinomas, melanomas, coloncancer, and other solid tumor cancers.

Yet another embodiment of the present invention provides methods for theisolation of and compositions comprising super-infective, tumor-specificmutants of the bacterium Mycobacterium avium and for their use in thediagnosis and treatment of sarcomas, carcinomas, melanomas, coloncancer, and other solid tumor cancers.

Yet another embodiment of the present invention provides methods forattenuation of parasite vector toxicity so as to reduce the risk ofseptic shock or other complications in the host, i.e., the patientreceiving vector-delivered gene therapy. Such methods includemutagenesis of parasites; isolation of parasite mutants with increasedtumor specificity, increased specificity for suicide gene expression andconcomitant reduced ability to infect normal host cells in the body;isolation of mutants with enhanced chemotactic abilities toward cancercell secretory products; isolation of mutants with genetically alteredlipopolysaccharide composition; and isolation of mutants with alteredvirulence genes so as to achieve specific survival of the parasiticvector in cancer cells as opposed to normal cells of the host body.

The present invention further encompasses use of microorganism vectorsfor diagnosis or treatment of solid tumor cancers.

The present invention may be understood more fully by reference to thefollowing definitions, detailed description of the invention,illustrative examples of specific embodiments and the appended figuresin which:

4. DEFINITIONS

Attenuation: Attenuation, in addition to its traditional definition inwhich a microorganism or vector is modified so that the microorganism orvector is less pathogenic, is intended to include also the modificationof a microorganism or vector so that a lower titer of that microorganismor vector can be administered to a patient and still achieve comparableresults as if one had administered a higher titer of the parentalmicroorganism or vector. The end result of attenuation is that the riskof toxicity as well as other side-effects is decreased, when themicroorganism or vector is administered to the patient.

Suicide gene: A suicide gene is defined as a gene that when delivered toa target cell and expressed by a vector of the present invention causesthe death of the target cell and/or the vector.

Super-infective: A super-infective vector is defined as a vector whichis able to attach and/or infect a target cell more readily as comparedto the wild type vector. Depending on the population density of theinoculum, the ratio between super-infective vectors and wild typevectors detectably infecting a target cell approaches 4:1, preferably30:1, more preferably 90:1. Most preferably, one is able to reduce theinoculum size and infection time so that only the super-infectivevectors have time to attach to and/or infect cancer cells growing incell culture in vitro or as tumors in vivo.

Tumor-specific: A tumor-specific vector is defined as a vector which isable to distinguish between a cancerous target cell and thenon-cancerous counterpart cell so that the vector preferentiallyattaches to, infects and/or remains viable in the cancerous target cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1 depicts a DNA cassette system for expressing pro-drugconverting enzymes. Each of the components is generated by PCR usingprimers dontaining specific restriction endonuclease sites NotI, NsiI,NcoI, SfiI or PacI that allow for simple interchange of individualcomponents. For example, (A) is the coding sequence for pro-drugconverting enzymes such as thymidine kinase, cytosine deaminase or humanmicrosomal p450 oxidoreductase; (B) is a promoter, which is active in aninducible, constitutive or cell specific manner; (C) is a N-terminalsecretion signal sequence, such as the β-lactamase signal sequence; and(D) is a C-terminal secretion signal sequence, such as theenteroinvasive E. coli hemolysin A signal sequence.

FIGS. 2A-B. FIGS. 2A-B are photomicrographs of Salmonella typhimuriumwild type strain ATCC No. 14028 infecting human melanoma cell line M2. Astarting population of ATCC No. 14028 was subjected to 10 cycles ofinfection into and recovery from M2 melanoma cells before use in theinfection assay shown in FIGS. 2A-B. FIG. 2A. Light micrograph of aninfected melanoma cell. FIG. 2B. DAPI staining of the cell showing cellnucleus, (n), and numerous bacteria inside the cell, (arrow).

FIGS. 3A-C. FIGS. 3A-C are photomicrographs of Salmonella typhimuriumwild type strain ATCC No. 14028 during the process of internalizationinto human melanoma cell line M2. FIG. 3A. Phase contrast micrograph ofa host cell. FIG. 3B. DAPI staining of the host cell showing theposition of the bacteria, (arrow), and the host cell nucleus, (n). FIG.3C. Lysosomal glycoprotein LAMP-1 antibody staining of the host cellshowing co-localization of the bacteria with lysosomes and/ormelanosomes.

FIGS. 4A-4F relate to producing converting enzyme expression constructsand expression using the same.

FIGS. 4A-B. Expression of the Herpes simplex thymidine kinase genecontaining a β-lactamase secretory signal sequence in Salmonellatyphimurium super-infective clone 72. FIG. 4A. Immunoblot analysis ofSalmonella typhimurium strains using an anti-TK monoclonal antibody.Lane 1: bacteria containing only the plasmid vector p279; Lane 2: strain14028 wt (CDC6516-60) (MO) containing the cytoplasmicly expressed formof TK (pHETK2); Lane 3: strain 14028 clone 72 containing thecytoplasmicly expressed form of TK (pHETK2); Lane 4: strain 14028 wt(MO) containing the β-lactamase fusion form of TK (p5-3); Lane 5: strain14028 clone 72 containing the β-lactamase fusion form of TK (p5-3); Lane6: strain 14028 wt (MO) containing the β-lactamase fusion form of TK(p2lA-2); Lane 7: strain 14028 clone 72 containing the β-lactamasefusion form of TK (p21A-2). Relative molecular mass×10³ is shown on theleft. No antibody reactivity is seen in the “vector only” control (Lane1). In each lane where the cytoplasmically expressed TK is present(Lanes 2 and 3) two major isoforms of the protein are seen; a highermolecular mass isoform containing a leader sequence and a lowermolecular mass isoform wherein the leader sequence has beenproteolytically cleaved off. In each lane where the bacteria express theTK gene β-lactamase signal sequence fusion (Lanes 4 to 7) two majorisoforms of the protein are also seen: a higher molecular mass formcontaining the signal sequence and a lower molecular mass isoformwherein the signal sequence has been proteolytically cleaved off, whichis the same apparent molecular mass as the processed form of thecytoplasmic enzyme.

FIG. 4B. Relative TK enzyme activity associated with each of the samplesin Figure A. Enzyme activity is expressed as the total number of countsof ¹²⁵IdC phosphorylated in a standard assay, Summers and Summers, 1977,J. Virol. 24:314-318. A small background is present in a bacterialextract from the vector only sample (Lane 1). Significantly higherlevels of TK activity are observed in the wild type and thesuper-infective clone 72 containing the cytoplasmic form of TK pHETK2,Lanes 2 and 3. Similar levels are observed in both the wild type andsuper-infective clone 72 containing the β-lactamase signal sequencefusion isoform of TK p5-3, Lanes 4 and 5. Lower levels are observed inboth the wild type and super-infective clone 72 containing theβ-lactamase signal sequence fusion isoform of TK p2lA-2, Lanes 6 and 7.

FIG. 4-C is a schematic of the different Herpes Simplex Virus thymidinekinase secretion and expression constructs.

FIG. 4-D is a schematic of the different human microsomal cytochromep450 oxidoreductase expression constructs.

FIG. 4-E is a schematic of the E. coli cytosine deaminase secretion andexpression construct.

FIG. 4-F is a graph showing the amount of 5-FC converted to 5-FU bydifferent bacteria.

FIGS. 5A-B. FIGS. 5A-B are photomicrographs of histologic sections froma Cloudman S91 melanoma/macrophage hybrid #48 growing subcutaneously ina DBA/2J mouse. The tumor was excised from a mouse that had beeninoculated 2 days earlier with 3×10⁵ c.f.u. Salmonella typhimuriumsuper-infective clone #72 carrying the HSV TK gene, clone #72⁵⁻³⁻². FIG.5A. A section stained with hematoxylin and eosin shows tumor cells witha central area of necrosis, denoted by arrows. FIG. 5B. A sectionstained with Brown-Brenn stain (tissue gram stain) shows gram negativebacteria in a necrotic area of the tumor, denoted by the arrow. Whenviewed under a light microscope, the bacteria stain pink/purple againsta yellow background.

FIG. 6. FIG. 6 is an electron micrograph of a section of a Cloudman S91melanoma/macrophage hybrid #48 tumor excised from a DBA/2J mouse thathad been inoculated i.p. 42 hours earlier with 4×10⁶ Salmonellatyphimurium super-infective clone 72. Visible in the micrograph are twoSalmonella typhimurium bacteria, denoted by arrows, along with numerousmelanosomes (m), sub-cellular organelles characteristic of melanomacells. The co-localization of Salmonella and such melanosomes indicatesthat the bacteria are present in the cytoplasm of the melanoma cell.Magnification=21,000×.

FIG. 7. FIG. 7 is a photomicrograph of a histologic section from aB16FlO melanoma growing subcutaneously in a C57BL/6J mouse. The tumorwas excised from a mouse that had been inoculated 42 hours earlier with1.8×10⁵ c.f.u. Salmonella typhimurium super-infective clone #72 carryingthe HSV TK gene. The sections were from the same tumor examined with theelectron microscope as detailed in FIG. 8. The section was stained withBrown-Brenn stain (tissue gram stain) and shows gram negative bacteriain a necrotic area of the tumor, denoted by arrows. When viewed under alight microscope, the bacteria stain pink/purple against a yellowbackground.

FIG. 8. FIG. 8 is an electron micrograph of a section from a B16FlOmelanoma tumor excised from a C57BL/6J mouse that had been inoculatedi.p. 42 hours earlier with 1.8×10⁵ Salmonella typhimuriumsuper-infective clone #72 carrying the HSV TK gene. The section was fromthe same tumor examined with the light microscope as detailed in FIG. 7.The micrograph shows numerous Salmonella typhimurium in extracellularspaces, denoted by arrows, and in an area of necrosis. A singlebacterium is also seen within the cytoplasm of a dying melanoma cell.The cytoplasm of the dying melanoma cell also contains numerous blackmelanosomes (m), characteristic of the B16FlO melanoma.Magnification=9,750×.

FIGS. 9A-D. FIGS. 9A-D depict growth of Cloudman S91 melanoma/macrophagehybrid #48 tumors in DBA/2J mice under various treatment conditions.Mice were inoculated s.c. in the flank region with 3×10⁵ melanoma cells.The tumors were palpable 8-10 days later, and some of the mice were thenfurther inoculated with Salmonella typhimurium super-infective clone #72carrying the HSV TK gene. Twenty-four hours post inoculation withbacteria, some groups of the mice were further inoculated i.p. with 2.0mg ganciclovir. Ganciclovir inoculations were repeated 6 times over a 5day period. Points represent caliper measurements of tumors in 2 to 5mice per treatment group at the days indicated. Measurements in mm weremade of length, width, and height for each tumor and volumes werecalculated in mm³. Average tumor volumes for each group of mice weredefined as 100% on day 0, the beginning day of treatment. (FIG. 9A).Control mice: no Salmonella; no ganciclovir (FIG. 9B). Ganciclovir only;(FIG. 9C). Salmonella only; (FIG. 9D). Salmonella+ganciclovir.

FIGS. 10A-B. FIG. 10A shows a control mouse and FIG. 10B shows aSalmonella typhimurium-infected (7B) DBA/2J mouse. The mice wereinoculated (s.c.) with Cloudman S91 melanoma/macrophage hybrid #48 tumorcells. Upon the appearance of palpable tumors some of the mice wereinoculated (i.p.) with 3×10⁵ c.f.u. Salmonella typhimurium clone 72containing the HSV TK gene (clone #72⁵⁻³⁻²), allowed to eat and drink adlibitum for 10 days, and then treated with Sulfatrim™ antibiotic intheir drinking water for several more days and photographed. Tumors inthe depicted mice were representative of the general state of tumorprogression in mice in Salmonella-treated and untreated cages.

FIGS. 11A-11H relate to the effects of gancicylovir on tumor cellgrowth, in vivo or in vitro.

FIGS. 11A-B. FIGS. 11A-B depict a control (FIG. 11A) and a Salmonellatyphimurium-infected (FIG. 11B) DBA/2J mouse. The mice were inoculated(s.c.) with Cloudman S91 melanoma/macrophage hybrid #48 tumor cells Uponthe appearance of palpable tumors, some of the mice were inoculated with3×10⁵ c.f.u. Salmonella typhimurium clone #72 containing the HSV TK gene(clone #72⁵⁻⁴⁻²). Control and Salmonella-infected mice were theninjected (i.p.) with 2.0 mg ganciclovir a total of 5 times over a 4 dayperiod. The mice were then treated with Sulfatrim™ antibiotic in theirdrinking water for several more days and photographed. The depicted miceare representative of the general state of tumor progression in mice,either in Salmonella-treated and untreated cages.

FIGS. 11(C-E) show the effect of ganciclovir on the growth of B16F10melanomas in mice with and without inoculation of Salmonella typhimuriumclone YS7211 (FIG. 11-1A); clone YS7213 (FIG. 11-1B); and clone YS7212(FIG. 11-1C).

FIG. 11-F is a graph showing the growth of B16F10 melanoma cells inmonolayer culture in the presence or absence of ganciclovir at 10 μg/mlor 25 μg/ml.

FIG. 11-G is a graph showing the effect of ganciclovir on the growth ofB16F10 melanomas in mice following inoculation of Salmonella typhimuriumclone YS7211 carrying the HSV thymidine kinase gene, YS7211/p5-3, withand without ganciclovir.

FIG. 11-H is a graph showing the effect of total amounts of gancicloviron the growth of B16F10 melanomas in mice following inoculation withSalmonella typhimurium clone YS7211 carrying the HSV thymidine kinasegene, YS7211/p5-3.

FIGS. 12A-B are electron micrographs. FIG. 12A is an electron micrographof a section from a HCT 116 human colon tumor excised from a BALB/cnu/nu mouse. The mouse had been inoculated i.p. 72 hours earlier with2.8×10⁵ c.f u. Salmonella typhimurium super-infective clone #72containing the HSV TK gene, clone #72⁵⁻³⁻². Shown in the micrograph arenumerous Salmonella typhimurium within the cytoplasm of a neutrophilassociated with the tumor. Some of the bacteria are undergoing divisionas denoted by arrows. The neutrophil or polymorphonucleoleukocyte ischaracterized by its multi-lobed nucleus (n), (Magnification=21,000×).

FIG. 12-B is an electron micrograph showing numerous Salmonellatyphimurium, denoted by arrows, in extracellular spaces as well ascontained within a single cell, possibly a neutrophil, seen in the upperleft. Also seen in the field are two unidentified cells that appear tobe dying as indicated by the large intracellular space, along withcellular debris.

FIGS. 13A-B. FIGS. 13A-B depict Leishmania amazonensis adhesion to humanmelanoma cell line M2. FIG. 13A. Phase contrast micrograph showingparasites attached to cell, (arrow). FIG. 13B. DAPI staining showing theparasite DNA, (arrows), and the host cell nucleus, (n).

FIGS. 14A-C. FIGS. 14A-C are photomicrographs of Leishmania amazonensisduring the process of internalization into human melanoma cell line M2.FIG. 14A. Phase contrast of a Leishmania trypomastigote, (arrow),entering a host cell. FIG. 14B. DAPI staining showing the position ofthe parasite, (arrow), and the host cell nucleus, (n). FIG. 14C.Lysosomal glycoprotein LAMP-1 antibody staining of the host cell showingco-localization of the bacteria and the lysosomes.

FIGS. 15 A-D. FIGS. 15 A-C are graphs showing growth of Salmonellatyphimurium clone 72 and clone YS7212 in minimal Medium 56 supplementedwith glucose only; Medium 56 with glucose plus adenine, vitamin B1,isoleucine, valine, and uracil; or Medium 56 with tumor extract (10%)only.

FIG. 15-D is a graph depicting growth of Salmonella typhimurium clones72 and YS7212 following invasion into human M2 melanoma cells inculture.

FIGS. 16A-D are graphs showing growth of B16F10 melanomas in C57B6 micewith and without inoculation of Salmonella typhimurium strains YS721(FIG. 16-A); YS7213 (FIG. 16-B); YS7211 (FIG. 16-C); and YS7212 (FIG.16-D).

FIG. 17 is a graph showing that combination of CD and 5-fluorocytosineprolong the survival of animals bearing B16F10 lung metastases when theanimals are infected with a tumor-specific vector carrying the cytosinedeaminase expression construct YS7212/pCD-Sec1.

FIG. 18 is a graph showing TNF-α production by human macrophagesincubated with lipopolysaccharide isolated from wild type and attenuatedstrains of Salmonella typhimurium.

FIG. 19 is a bar graph demonstrating the effect of clones YS7211 andYS7212 expressing the HSV thymidine kinase gene, YS7211/p5-3 andYS7212/p5-3, respectively, on mice bearing metastatic B16F10 tumors withor without ganciclovir treatment.

6. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the isolation of novel therapeuticand diagnostic parasitic vectors for solid tumor cancers, such assarcomas, carcinomas, lymphomas or other solid tumor cancers, forexample, germ line tumors and tumors of the central nervous system,including, but not limited to, breast cancer, prostate cancer, cervicalcancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer,thyroid cancer, astrocytoma, glioma, pancreatic cancer, stomach cancer,liver cancer, colon cancer, melanoma and their use. Described, in detailbelow, are the novel intracellular parasite vectors; methods for theisolation of the novel vectors; genetic engineering of the isolatedvectors; and methods for use of the novel vectors as well as othervectors in treatment or detection of solid malignant tumors, includingmetastatic tumors and tumor cells.

6.1. Novel Vectors and Methods For Their Isolation

The isolated vectors, which are for example, bacteria, fungi orprotista, are able to differentiate between cancerous cells andnon-cancerous counterpart cells. For example, the isolated vectors areable to differentiate melanoma cells from melanocytes or differentiatecolon cancer cells from normal colon epithelial cells. Table I is arepresentative list, which is in no way meant to limit the presentinvention, of intracellular parasitic and pathogenic microorganismswhich are useful as tumor-specific vectors for the present inventionand/or for isolation of novel mutant strains which are super-infectiveand tumor-specific vectors for use in the present invention.

TABLE 1 REPRESENTATIVE LIST OF ORGANISMS USEFUL AS VECTORS Gram negativebacteria Borrelia burgdorferi Brucella melitensis Escherichia colienteroinvasive Escherichia coli Legionella pneumophila Salmonella typhiSalmonella typhimurium Shigella spp. Treponema pallidum Yersiniaenterocohtica Gram positive bacteria BCG (Bacillus Calmette-Guerin)Chlamydia trachomatis Listeria monocytogenies Mycobacterium aviumMycobacterium bovis Mycobacterium tuberculosis Mycoplasma hominisRickettsiae quintana Streptococcus spp. Fungi Cryptococcus neoformansHistoplasina capsulatum Pneumocystis carnii Apicomplexans Eimeriaacervulina Neospora caninum Plasmodium falciparum Sarcocystis suihominisToxoplasma gondii Kinetoplastida Leishinania amazonensis Leishmaniamajor Leishmania mexacana Leptomonas karyophilus Phytomonas spp.Trypanasoma cruzi Microsporidians Encephahtozoon cuniculi Nosemahelminthorum Unikaryon legeri

The bacterium Salmonella typhimurium, the bacterium Mycobacterium avium,and the protozoan Leishmania amazonensis are each particularly usefulvectors for the present invention, since each of these organisms showsnatural preference for attachment to and penetration into certain solidtumor cancer cells in tissue culture, as opposed to non-cancerouscounterpart cells. Since these vectors, such as Salmonella, have anatural ability to distinguish between cancerous cells and theirnon-cancerous counterpart cells they are directly applicable to themethods for diagnosis or treatment according to the present invention.However, this tumor-specific ability, as well as, the ability to besuper-infective as compared to the “wild type” parent strain may beenhanced and selected for by using the methods of the present inventiondescribed in Sections 6.1.1-6.1.4., infra.

6.1.1. Isolation By Cycling Through In Vitro Tissue Culture

One embodiment of the present invention is to isolate the novel vectorsof the present invention by cycling a microorganism through apre-selected target cell, preferably a solid tumor cancer cell, with oneor more cycles of infection in in vitro tissue culture so that thecycled population and/or clonal isolates therefrom demonstrate enhancedinfectivity of the target tumor cell as compared to the startingmicrobial population and enhanced selectivity as compared to thenon-cancerous counterpart cell. The method entails selecting a parasiteor pathogen and adding the microorganism to an in vitro tissue culturesystem of the particular type of solid tumor that one wishes to use as atarget cell. For example, if one desires to target melanoma tumors, thetarget cell may be M2 human melanoma cells. After incubating the tumorcells and microorganisms together, which allows enough time for themicroorganism to attach and/or infect the tumor cell, the tumor cellculture is washed with either buffer or medium which contains anantibiotic agent effective against the specific microorganism used. Theantibiotic agent kills any microorganisms that have not attached to andinfected the tumor cell. If desired, the infected tumor cell culture maybe incubated further in medium containing antibiotic for varying times,depending on the type of population of microorganisms to be isolated.For example, for longer incubation times, the microorganism populationisolated has enhanced survival and/or proliferative abilities inside thetumor cells as compared to the starting population of microorganisms.Additionally, the isolated populations can be cultured to isolate singlecolony clones using standard techniques.

The infected animal cells are collected and lysed, thus freeing theinternalized microorganisms. The microorganism can then be isolated, forexample, by centrifugation (2000×g for 4 minutes) and resuspending infresh medium. The isolated microbial population may then be used foradditional cycles of infection into and isolation out of the targettumor cell. The isolated microbial population may be placed first inappropriate growth medium for 1-2 doubling times before being subjectedto additional infection cycles to insure their viability. The isolatedmicrobial population may also be cultured so as to isolate and collectsingle colony clones. The isolated microorganisms may also undergo knownin vitro techniques to determine their relative infective and selectiveabilities as compared to the “wild type” parent strain which did notundergo in vitro selection. For example, in side by side comparisons onemay test the relative infectiveness of the isolated microorganism ascompared to the “wild type” by using assays designed to quantitate thenumber of microorganisms which have attached to or invaded the targettumor cell and/or their ability to distinguish between cancerous andnon-cancerous cells. In addition, parameters such as microorganismpopulation density, may be varied in these in vitro assays which assistsin determining what effect the overall concentration of inoculum of theclone or population being tested has on the ability of themicroorganisms to differentiate between the target tumor cells and theirnon-cancerous counterparts.

For an illustrative example of super-infective, tumor-specific vectorsisolated by cycling through in vitro tissue culture, see Section 7,infra.

6.1.2. Isolation By Cycling Through In Vivo Solid Tumors

Another embodiment of the present invention is to isolate the novelvectors of the present invention by cycling the microorganism throughsolid tumors in vivo. This procedure is performed using experimentaltumor models in mammals such as, for example and not by way oflimitation, B16 mouse melanoma cells which form melanoma tumors in C57B6mice and HCT116 human colon carcinoma cell which form colon carcinomasin nu/nu and other immuno-compromised mice. Additionally, fresh biopsiesof tumor tissue which are obtained surgically from a cancer patient maybe used to inoculate nu/nu, scid or other immuno-compromised mice. Thesetumors in mice which have grown from inoculated cancer cells are used asin vivo targets for the isolation of super-infective and tumor-specificvectors in a similar manner as in vitro target cells. Any tumor growingin mice or any other animal may be used in the present invention as atarget for the isolation of super-infective and tumor-specific vectorsin vivo.

Once the tumor is established in the mouse, by, for example, inoculationof cancer cells sub-cutaneously or transplantation of a tumor mass, theselected microorganism is inoculated into the mouse. After apre-determined infection time after inoculation in which themicroorganism becomes co-localized with the tumor and/or infects thetumor cells, the mice are sacrificed, the tumors excised, weighed andhomogenized. An aliquot may be diluted into the proper microorganismgrowth medium and incubated at the proper growth conditions for 1-2population doublings to insure the recovery of viable microorganisms forsuccessive inoculations into tumor bearing mice. Further, if theisolated population is to undergo successive inoculations in tumorbearing mice, upon each successive inoculation, the number ofmicroorganisms in the inoculate and the time of infection may be reducedto increase the stringency of selection for tumor-specific isolates.Additionally, the isolated populations can be cultured to isolate singlecolony clones using standard techniques. The isolated microorganisms maybe used also in in vitro assays to determine their relative infectiveand selective abilities as compared to the “wild type” parent strainwhich did not undergo an in vivo selection procedure.

For an illustrative example of super-infective, tumor-specific vectorsisolated in vivo in tumor-bearing mice, see Section 9, infra.

6.1.3. Isolation By In Vitro Chemotaxis Using Medium Conditioned By theTarget Tumor Cell

Another embodiment of the present invention is to provide methods forisolating super-infective and/or tumor cell-specific vectors bychemotaxis so that the isolated microorganisms have increasedchemotactic ability towards tumor cell secretory products. The methodentails using capillary tubes which are loaded with either liquidcontrol medium or medium that has been conditioned by the target tumorcell as described by Adler (1973, J. General Microbiology 74:77-91).Conditioned medium is medium in which the target cells have been grownand subsequently has been filtered to remove the cells. One end of thecapillary is sealed in a flame; the capillary is then quickly passedseveral times through a flame and is immediately plunged open end downinto a beaker containing either the conditioned or control medium. Asthe capillaries cool, the liquid is drawn up inside.

The loaded capillary tubes are inserted open end down into a centrifugetube containing medium and a suspension of the pre-selectedmicroorganism. After a pre-determined period of incubation at 37° C. inwhich the microorganism chemotacts into the capillary tubes, thecapillary tubes are removed with forceps, the sealed ends are opened andthe opened capillaries are transferred into centrifuge tubes containingnutrient medium appropriate for the particular microorganism. It isimportant that the upper tips of the capillary tubes are covered with anappropriate medium for the particular type of microorganism to assurequantitative recovery of the microorganism from the capillary tubesduring centrifugation. The capillary tubes are centrifuged, for example,at 4000×g for 4 minutes, to force the microorganism out of the tube. Thecapillary tubes are removed, the microorganism resuspended, and analiquot spread onto the appropriate medium in either solid or liquidform to allow for quantitation.

Significant increases in the number of microorganisms entering into thecapillary tubes containing conditioned medium as compared to controlsindicates a positive chemotactic response toward secreted products ofthe target cell found in the conditioned medium. The populationsisolated by this in vitro technique can undergo successive chemotaxisassay isolation or be used to isolate single colony clones. These clonesor populations can be compared to the “wild type” parent strain in theirability to distinguish between the target cancerous cell and thenon-cancerous counterpart cell as well as for super-infective ability.

For an illustrative example of super-infective, tumor-specific vectorsisolated by in vitro chemotaxis using tumor cell-conditioned medium, seeSection 8, infra.

6.1.4. Isolation of Mutagenized Vectors

In any of the above-described methods for isolating super-infective,tumor-specific microorganisms, the “wild type” parent microorganism canbe subjected first to mutagenesis before the microorganism is subjectedto any isolation or selection procedure of the present invention. Forexample, bacteria are subjected to treatment with nitrosoguanidine andultraviolet B irradiation so that the hereditary genetic material ismodified resulting in the altered expression of genes, bothqualitatively and quantitatively, in the microorganism. Other types ofchemical and high-energy mutagenesis are well known in the art. Forexample, alkylating agents such as dimethyl nitrosamine or ethyl methanesulfonate, or intercalating agents, such as ethidium bromide. Otherapproaches include transposon mutagenesis to introduce genetic flocks orfusions of genes with new promoters. Any mutagen can be used in thepresent invention to create mutant strains of microorganisms which maythen undergo any of the selection methods of the present invention.

For an illustrative example of mutagenesis, see Section 7.1, infra.

6.2. Genetic Manipulation of the Selected Vectors For Delivery of Genesand/Or Gene Products To the Target Solid Tumor Cells as Well as ForAttenuation of Virulence 6.2.1. Genetic Manipulation For Delivery ofGenes and/Or Gene Products To the Target Site

After the selection processes described above in which one obtains asuper-infective, tumor-specific vector, one can genetically engineersuch vectors so that any desired gene or gene product is delivered to atarget site, preferably the site of a solid tumor, more preferably, intothe tumor cell itself, the necrotic areas of the tumor or intotumor-associated lymphocytes and macrophages. Additionally, one cangenetically alter naturally occurring microorganisms which have anatural ability to infect tumor cells and/or be tumor-cell specific.These vectors are genetically engineered by a wide variety of methodsknown in the art, for example, transformation or electroporation. In apreferred embodiment of the present invention the vectors are engineeredto deliver suicide genes to the target tumor cells. These suicide genesinclude pro-drug converting enzymes, such as Herpes simplex thymidinekinase (TK) and bacterial cytosine deaminase (CD). TK phosphorylates thenon-toxic substrates acyclovir and ganciclovir, rendering them toxic viatheir incorporation into genomic DNA. CD converts the non-toxic5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), which is toxic viaits incorporation into RNA. Additional examples of pro-drug convertingenzymes encompassed by the present invention include cytochrome p450NADPH oxidoreductase which acts upon mitomycin C and porfiromycin(Murray et al., 1994, J. Pharmacol. Exp. Therapeut. 270:645-649).

Prodrug converting enzymes are being widely employed for use in genetherapy of malignant cancers (Vile and Hart, 1993, Cancer Res.53:3860-3864; Moolten and Wells, 1990, J. Natl. Cancer Inst. 82:297-300;Wagner, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1441-1445; Mullen,1994, Cancer Res. 54:1503-1506; Huber et al., 1993, Cancer Res.53:4619-4625; Waldman et al., 1983, J. Biol. Chem. 258:11571-11575;Mullen, et al., 1992, Proc. Natl. Acad. Sci. 89:33-37; Austin and Huber,1993, Mol. Pharmacol. 43:380-387). Table 2 is an illustrative list ofpro-drug converting enzymes (Bagshawe, 1995, Drug Dev. Res. 34:220-230).

Prodrug converting enzymes have been expressed in several bacteria. TheHerpes simplex virus has been expressed in E. coli (Garapin, 1980, Proc.Natl. Acad. Sci. USA 78:815-819; Waldman et al., 1983, J. Biol. Chem.258:11571-11575). Similarly, Simula et al., 1993, Toxicology 82:3-20,expressed the prodrug converting enzyme cytochrome p450 oxidoreductasein Salmonella typhimurium which confered sensitivity to mitomycin.

TABLE 2 REPRESENTATIVE PRO-DRUG CONVERTING ENZYMES FOR USE IN VECTORTHERAPY Enzyme Pro-drug Reference Carboxypeptidase G2 benzoic acidmustards Bashawe et al., 1988; Springer et al., 1990 aniline mustardsDavies et al., 1994 phenol mustards Springer et al. Beta-glucuronidasep-hydroxyaniline Roffer et al., 1991 mustard glucuronideepirubicin-glucuronide Halsma et al., 1992 Mitaku et al., 1994Penicillin-V-amidase adriamycin-N Kerr et al., 1990 phenoxyacerylPenicillin-G-amidase N-(4′-hydroxyphenyl- Bignami et al., 1992acetyl)-palytoxin doxorubicin melphalan Vrudhula et al., 1993β-lactamase nitrogen mustard- Alexander et al., 1991 cephalosporinβ-phenylenediamine vinblastine derivative- cephalosporin cephalosporinmustard Meyer et al., 1993 Svensson et al, 1993 β-glucosidasecyanophenylmethyl-β-D- Rowlandson-Busza gluco-pyranosiduronic et al.,1991 acid Nitroreductase 5-(adaridin-1-yl-)2, 4- Knox et al., 1988;dinitrobenzamide Somani and Wilman, 1994 Carboxypeptidase Amethotrexate-alanine Haenseler et al., 1992 Bagshawe et al., 1988, Br.J. Cancer 58:700-703. Springer et al., 1990, J. Med. Chem. 33:677-681.Davies et al., 1994, Ann. Oncol. 5 (Suppl 5):73(abstr). Springer et al.,1994, A novel bisiodo-phenol mustard in antibody-directed enzymepro-drug therapy (ADEPT). In: Programme of Eleventh HammersmithConference. Advances in the Application of Monoclonal Antibodies.London: Hammersmith Hospital (abstr). Haisma et al., 1992(a), CancerImmunol. Immunother. 34:343-348. Roffler et al., 1991, Biochem.Pharmacol. 42:2062-2065. Haisma et al., 1992(b), Br. J. Cancer88:474-478. Mitaku et al., 1994, Ann. Oncol. 5 (Suppl 5):76 (abstr).Kerr et al., 1990, Cancer Immunol. Immunother. 31 :202-206. Bignami etal., 1992, Cancer Res. 52:5759-5764. Vrudhula et al., 1993, J. Med.Chem. 38:919-923. Alexander et al., 1991, Tetrahedron Lett.32:3296-3272. Meyer et al., 1993, Cancer Res. 53:3956-3963. Svensson etal., 1992, Bioconj. Chem. 3:176-181. Rowlandson-Busza et al., 1991,Cytotoxicity following specific activation of amygladin. In: MonoclonalAntibodies, Epenetos AA (ed), London: Chapman & Hall, pp. 179-183. Knoxet al., 1988, Biochem. Pharmacol. 41:4661-4669. Somani et al.; 1994,Ann. Oncol. 5 (Suppl 5):73 (abstr). Haenseler, E., Esswein, A., Vitols,K. S., Montejano, V., Mueller et al., 1992, Biochemistry 31 :214-220.

However, pro-drug converting enzymes such as the TK and CD enzymes, whensynthesized in bacteria such as Salmonella or Escherichia coli, are notnormally secreted from the bacteria. Accordingly, the expressionconstruct is designed such that the microorganism-produced gene productsare secreted by the microorganism. Thus, TK or CD are able to generatephosphorylated acyclovir, ganciclovir, or 5-FU, within the target tumorcell cytoplasm and interstitial spaces of the target tumor. Secretion ofTK and CD is achieved by introducing into the expression construct asecretory signal sequence, for example, from the β-lactamase gene(Talmadge et al., 1980, Proc. Natl. Acad. Sci. USA 77:3369-3373).

Alternate signal sequences, in addition to β-lactamase, are alsoencompassed by the present invention. Bacteria, for example, are knownto have several means for secretion into the periplasm and the outsidemedia. The most typical secretion sequences are N-terminal signalsequences containing hydrophobic transmembrane spanning domains. Thesesequences serve to guide the protein through the membrane and areremoved as- or after the protein crosses the membrane. Prokaryotic andeukaryotic N-terminal signal sequences are similar and it has been shownthat eukaryotic N-terminal signal sequences are capable of functioningas secretion sequences in bacteria. In a preferred embodiment, the geneencoding the enzyme β-lactamase (penicillinase) is used as the source ofthe signal sequence. This signal sequence is a well studied example of abacterial enzyme which is secreted both into the periplasm and into theexternal media.

Further, some bacterial proteins utilize a different secretion signalwhich is located at the C-terminus. The enteroinvasive E. coli hemolysinA (hlyA) is the best studied member of this group. It has been shownthat the secretion signal is present in the last 60 amino acids of thatprotein and that transfer of this domain to other proteins can result intheir direct secretion into the media when the accessory proteins fromthe hemolysin operon (hylC, A, B, & D) are present (Su et al., 1992,Microbial Pathogen. 13:465-476). An illustrative list of secretedproteins reviewed by Pugsley is presented in Table 3, (Pugsley A. P.,1988, Protein secretion across the outer membrane of gram-negativebacteria. In: Protein Transfer and organelle Biogenesis, R. C. Dand andP. W. Robbins (eds), Academic Press, Inc., Harcourt Brace Jovanovich,Publishers, San Diego, pp 607-652).

TABLE 3 SOURCES OF SECRETION SIGNALS FOR PRO-CONVERTING ENZYMES Locationin Transfected Ref. Protein Organism E. coli and type of signal No.Chitinase Serratia marcescens released into medium N- 14 terminal signalα-Hemolysin E. coli released into medium C- 9 terminal signal Heatlabile various E. coli Similar to cholera toxin 2 12, 7, enterotoxin Istrains subunits (A&B); N- 29 terminal signal in both; primarily inperiplasm Heat-stable various E. coli N-terminal signal 11, 28enterotoxin I strains peptide; secreted into the media Heat-labilevarious E. coli N-terminal signal peptide 17 enterotoxin II strainsPullulanase Kelbsiella Release into the medium; 3, 27, pneumoniaeN-terminal signal peptide 6 Serine protease S. marcencens Secreted intothe 31 medium; N-terminal signal peptide Pectate lyase Erwinia Mainly inthe periplasm 15, 5 chrysanthemi Pectate lyase E. carotovara Periplasm18, 32 Protease E. chrysanthemi Secreted into the medium 30, 1 AerolysinAeromonas Periplasm, N-terminal 13 hydrophila signal sequence(processed) Phospholipase Pseudomonas not secreted by E. coli N- 4, 19,C aeruginosa terminal signal sequence 26 Exotoxin A P. aeruginosa notsecreted by E. coli 10 Cholera toxin Vibrio cholerae Mainly periplasmic;25, 20 2 subunits Hemolysin V. cholerae Periplasm 21 DNase V. CholeraePeriplasm 22, 8 Thermostable V. parahaemolyticus Periplasm N-terminal 24Hemolysin signal peptide IgA protease Haemolphilis Periplasm 2influenzae IgA protease Nisseria Secreted into the medium 16 gonorrhoeaePertussis toxin Bordetella Periplasm; 5 subunits all 23 pertussis withN-terminal signal peptides 1. Barras et al., 1986, FEMS Microbiol. Lett.34:343-348. 2. Bricker et al., 1983, Proc. Natl. Acad. Sci. USA80:2681-2685. 3. Chapon et al., 1985, J. Bacteriol. 164:639-645. 4.Coleman et al., 1983, J. Bacteriol. 153:909-915. 5. Collmer et al.,1985, J. Bacteriol. 161:913-920. 6. d'Enfert et al., 1987, EMBO J.6:3531-3538. 7. DaRas et al., 1980, Nature 288:499-501. 8. Focareta etal., 1985, FEMS Mcrobiol. Lett. 29:161-166. 9. Goebel et al., 1984,Structure, function and regulation of the plasmid-encoded hemolysindeterminant of E. coli. In Plasmids in Bacteria, D. R. Hehnski, S. N.Cohen, D. B. Cloewell, D. A. Jackson, and A. Hollaender (eds), pp.791-805, Plenum, New York. 10 . Gray et al., 1984, Proc. Natl. Acad.Sci. USA 81:2645-2649. 11. Guzmin-Verduzco et al., 1983, J. Bacteriol.154:146-151. 12. Hirst et al., 1984, Proc. Natl. Acad. Sci. USA81:7752-7756. 13. Howard et al., 1986, Mol. Gen. Genet. 204:289-295. 14.Jones et al., 1986, EMBO J. 5:2377-2383. 15. Keen et al., 1984, J.Bacteriol. 159:825-831. 16. Koomey et al., 1982, Proc. Natl. Acad. Sci.USA 79:7881-7885. 17. Lee et al., 1983, Infect. Immun. 42:264-268. 18.Lei et al., 1985, Gene 35:63-70. 19. Lory et al., 1983, Gene 22:95-101.20. Mekalanos et al., 1983, Nature 306:551-557. 21. Mercuric et al.,1985, Mol. Gen. Genet. 200:472-475. 22. Newland et al., 1985, Infect.Immun. 47:691-696. 23. Nicosia et al., 1986, Proc. Natl. Acad. Sci. USA83:4631-4635. 24. Nishibuchi et al., 1985, J. Bacteriol. 162:558-564.25. Pearson et al., 1982, Proc. Natl. Acad. Sci. USA 79:2976-2980. 26.Pritchard et al., 1986, J. Bacteriol. 167:291-298. 27. Pugsley, A. P.,1988, Protein secretion across the outer membrane of gram-negativebacteria. In: Protein Transfer and Organelle Biogenesis, R. C. Dand andP. W. Robbins (eds), Academic Press, Inc., Harcourt Brace Jovanovich,Publishers, San Diego, pp 607-652. 28. So et al., 1980, Proc. Natl.Acad. Sci. USA 77:4011-4015. 29. Spicer et al., 1982, J. Biol. Chem.257:5716-5721. 30. Wandersman C. (unpublished results cited in Pugsley,1988). 31. Yanigida et al., 1986, J. Bacteriol. 166:937-944. 32. Zink etal., 1985, Appl. Environ. Microbiol. 49:714-717.

In another embodiment of the present invention, the desired genesexpressed from the expression constructs are under the specificregulatory control of certain types of promoters. These promoters may beeither constitutive, in which the genes are continually expressed,inducible, in which the genes are expressed only upon the presence of aninducer molecule(s) or cell-type specific control, in which the genes,including but not limited to suicide genes, are expressed only incertain cell types. Further, expression of foreign genes includingprodrug converting enzymes frequently alters the phenotype of thebacteria. Therefore, it would be an advantage to drive the expression ofa prodrug converting enzyme under exogenous control. This would allowexploitation of the bacterial phenotypes such as tumor targeting andamplification, after which time it would be beneficial to express theprodrug enzyme. Inducible promoters drive gene expression under specificconditions. Furthermore, exogenously inducible promoters respond tospecific stimuli including chemical signals which can be artificiallyintroduced. It would be an advantage to drive the expression of aprodrug enzyme using an exogenously introduced agent which is approvedfor use in humans. The “SOS” response of bacteria (Friedberg et al., In:DNA Repair and Mutagenesis, pp. 407-455, Am. Soc. Microbiol. Press,1995) is a response inducible by numerous agents includingchemotherapeutic alkylating agents such as mitomycin (Oda et al., 1985,Mutation Research 147:219-229; Nakamura.et al., 1987, Mutation Res.192:239-246; Shimda et al., 1994, Carcinogenesis 15:2523-2529) which isapproved for use in humans. Promoter elements which belong to this groupinclude umuC, sulA and others (Shinagawa et al., 1983, Gene 23:167-174;Schnarr et al., 1991, Biochemie 73:423-431). The sulA promoter includesthe ATG of the sulA gene and the following 27 nucleotides as well as 70nucleotides upstream of the ATG (Cole, 1983, Mol. Gen. Genet.189:400-404). Therefore, it is useful both in expressing foreign genesand in creating gene fusions for sequences lacking initiating codons.

In one embodiment, for example, the expression of the gene is controlledby a bacterial promoter which is activated in specific target cells. Ina preferred mode of this embodiment, the bacterial promoter is activatedprimarily in specific target cells. In another embodiment, for example,the expression of the gene is controlled by a bacterial promoter whichis activated only in specific tumor cells. An illustrative example of anexpression construct which expresses a gene under the control of apromoter with the necessary secretion signal sequence is diagrammed inFIG. 1.

In a preferred embodiment of the present invention, the expression ofthe gene is under the control of a promoter which is active only in thetarget cell. Microorganism promoters that are specifically orpreferentially active in a target tumor cell are isolated by a number ofdifferent methods. For example, one method is using IVET (in vivoexpression technology) promoter trap procedure for isolatingspecifically induced genes. This procedure is carried out by taking, forexample, a random pool of Salmonella typhimurium DNA insertionsgenerated by Sau3A restriction enzyme and cloning the fragments into thepromoter trap vector pIVET (Slauch et al., 1994, Methods Enzymol.235:481-492; Mahan et al., 1993, Science 259:686-688). The cloning siteis at the position of the promoter for the purA gene which is requiredfor the synthesis of cyclic AMP. This representative pool is transfectedback into Salmonella typhimurium and an integration event is inducedwhich results in replacement of the endogenous purA gene. The populationof bacteria carrying an integrated IVET plasmid is allowed to infect ananimal bearing a solid tumor of the cell type of choice and after 24hours bacteria are isolated from the tumor. Only those bacteria thatreceived a plasmid whose random piece of Sau3A restricted DNA acts as apromoter within the tumor cells is capable of surviving. In addition tocontrolling the transcription of purA, the Sau3A restricted DNA promoteralso controls the expression of the β-galactosidase gene. Two types ofpromoters are isolated which allow the survival of bacteria withintumors, constitutive and regulated. The constitutive promoter continuesto control the positive expression of both genes, inside and outside ofthe tumor. Whereas the regulated promoter is no longer active in cellsother than the target cell.

Another method for identifying promoters that are active in tumors is toidentify tumor-specifically induced microbiological gene products usingtwo dimensional gel electrophoresis. For example, to determine whichgene products are specifically or preferentially expressed in melanomacells rather than macrophages, the method entails three parallelinfections which proceed in tandem: (1) 5×10⁷ clonal microorganisms areallowed to infect 5×10⁶ melanoma cells, (2) 5×10⁷ clonal microorganismsare allowed to infect 5×10⁶ macrophages, and (3) 5×10⁷ clonalmicroorganisms are maintained in growth phase in LB broth. After a 30minute infection the cells are washed with DMEM with 10 μg/ml gentamicin(for melanoma cells), RPMI 1640 with 10 μg/ml gentamicin (formacrophages) and LB without gentamicin (for free microorganisms). Aftertwo hours the cells are pretreated with 50 mg/ml cyclohexamide toinhibit host cell protein synthesis for 15 minutes. The cells are thenwashed and placed in labeling medium (minus methionine) containing 75μCi/ml ³⁵S-methionine for 30 minutes, followed by 1 hour in normalmedium. The cells are then harvested, denatured in 7M urea buffer andsubjected to isoelectrofocusing (IEF) followed by sodium dodecylsulfate(SDS) polyacrylamide gel electrophoresis (PAGE) and analysis byautoradiography. Gene products specifically expressed in melanoma cellsappear as protein spots from microorganism-infected melanomas but notfrom microorganism-infected macrophages or from free microorganisms. Themicroorganismal genes that are specifically expressed are cloned from aλgt11 expression library using antiserum prepared from proteins derivedfrom preparative IEF and SDS-PAGE gels. Subsequent cloning from a cosmidlibrary results in DNA fragments containing the promoter elements forthe tumor-specific expressed gene product.

Yet another method for isolating promoters which are specifically orpreferentially activated in the target tumor cells is transposonmutagenesis. Transposon mutagenesis results in a pool of random mutantswhich can be tested for their ability to survive in epithelial cells butnot in target tumor cells. Mutants are first tested for their continuedability to persist in epithelial cells. Mutants no longer able tosurvive will be selected against. Surviving mutants are picked at randomand placed in a numbered array using 96 well plates. The target tumorcells are grown in 96 well plates and individually infected with amicroorganism at a microorganism to host ratio of about 10:1 for 30minutes, followed by washing and treatment with 10 μg/ml gentamicin.After 24 hours the plates are rinsed and stained with 0.4% trypan blueto determine the ratio of living cells(clear) to dead cells (blue) usinga 96 well plate reader. Microorganisms which are unable to survivewithin the target tumor cell are recovered from the original numberedplate. The genes are then cloned using the transposon as a geneticmarker to isolate the DNA containing the tumor-specific expressed geneand its promoter.

The vectors which can express the various pro-drug or “suicide genes”when given to the host should not confer antibiotic resistance to thehost and more importantly the bacteria should remain as sensitive to asmany antibiotics as possible. Therefore, these vectors should not carryany antibiotic resistance markers. This can pose a problem inmaintaining the expression vectors in the bacteria in absence ofselective pressure. However, the are a number of methods in which thevectors can be stably maintained without resorting to antibioticresistance. For example, one such method is the construction ofchromosomally integrated vectors expressing pro-drug converting enzymesor other “suicide genes” as described by Donnenberg, 1991, Am. Soc.Microbiol., Annual Meeting, Abstract B-111, p.4; Donnenberg and Kaper,1991, Infect. Immun. 59:4310-4317; and Ried and Collmer, 1987, Gene57:239-246. Another method is the construction of stable episomalplasmids encoding “suicide genes” or pro-drug converting enzymes using abalanced lethal system. Such balanced lethal systems are defined by thefact that the vector encodes for a function that compensates for adeficiency in the bacteria, such that the presence of the vector isessential for the survival of the bacteria. Such a system is describedby Galan et al., 1990, Gene 94:29-35. This system has the advantage overchromosomal integration in that the plasmids are multicopy and,therefore, achieve higher expression levels.

6.2.2. Genetic Manipulation For Attenuation of Virulence

Many of the microorganisms encompassed by the present invention arecausative agents of diseases in humans and animals. For example, sepsisfrom gram negative bacteria is a serious problem because of the highmortality rate associated with the onset of septic shock (R. C. Bone,1993, Clinical Microbiol. Revs. 6:57-68). Therefore, to allow the safeuse of these vectors in both diagnostics and treatment of humans andanimals, the microorganism vectors are attenuated in their virulence forcausing disease. In the present invention, attenuation, in addition toits traditional definition in which a microorganism or vector ismodified so that the microorganism or vector is less pathogenic, isintended to include also the modification of a microorganism or vectorso that a lower titer of that derived microorganism or vector can beadministered to a patient and still achieve comparable results as if onehad administered a higher titer of the parental microorganism or vector.The end result is to reduce the risk of toxic shock or other sideeffects due to administration of the vector to the patient. Suchattenuated microorganisms are isolated in a number of techniques. Suchmethods include use of antibiotic-sensitive strains of microorganisms,mutagenesis of the microorganisms, selection for tumor-specific,super-infective microorganism mutants in culture or in tumor-bearinganimals, selection for microorganism mutants that lack virulence factorsnecessary for survival in normal cells, including macrophages andneutrophils, and construction of new strains of microorganisms withaltered cell wall lipopolysaccharides. For example, in Section 6.1 etseq. where methods are described for the isolation of super-infective,tumor-specific vectors, these same methods are also methods forisolating attenuated vectors; super-infective, tumor cell-specificvectors are by definition attenuated. As the vectors are highly specificand super-infective, the difference between the number of infectingbacteria found at the target tumor cell as compared to the non-cancerouscounterparts becomes larger and larger as the dilution of themicroorganism culture is increased such that lower titers ofmicroorganism vectors can be used with positive results.

Further, the microorganisms can be attenuated by the deletion ordisruption of DNA sequences which encode for virulence factors whichinsure survival of the microorganisms in the host cell, especiallymacrophages and neutrophils, by, for example, homologous recombinationtechniques and chemical or transposon mutagenesis. For example, a numberof these virulence factors have been identified in Salmonella. Many, butnot all, of these studied virulence factors are associated with survivalin macrophages such that these factors are specifically expressed withinmacrophages due to stress, for example, acidification, or are used toinduced specific host cell responses, for example, macropinocytosis,Fields et al., 1986, Proc. Natl. Acad. Sci. USA 83:5189-5193. Table 4 isan illustrative list of Salmonella virulence factors which, if deletedby homologous recombination techniques or chemical or transposonmutagenesis, result in attenuated Salmonella.

TABLE 4 REPRESENTATIVE VIRULENCE FACTORS FOR SALMONELLA TYPHIMURIUM ANDOTHER BACTERIA Virulence Factor or Loci, Specific Stress Overcome orStimulated Response Reference Acidification Alpuche-Aranda et al., 19925′-adenosine monophosphate Biochenko and Levashev, 1987 Cytolysin Libbeyet al., 1994 Defensin resistance loci Fields et al., 1989 DNAK Buchmeierand Hefferon, 1990 Fimbriae Ernst et al., 1990 GroEL Buchmeier andHefferon, 1990 Induced Macropinocytosis Alpuche Aranda, et al., 1994Ginocchio et al., 1992 Jones et al., 1993 Inv loci Betts and Finlay,1992 Galon and Curtis III Ginocchio et al., 1992 Lipoprotein Stone etal., 1992 LPS Gianeiella et al., 1973 Stone et al., 1992 Lysosomalfusion inhibition Ishibashi et al., 1992 Macropage survival lociFieldsetal., 1989 Oxidative stress (Sox; in E. coli) Nunoshiba et al.,1993 PhoP and PhoQ Behlau and Miller, 1993 Groisman et al., 1993 Milleret al., 1989 Pho activated genes (pag; e.g., pagB Abshire and Neidhardt,1993 and pagC) Hefferon et al., 1992 Miller et al., 1992 Miller et al.,1989 Pulkkinen and Miller, 1991 Stone et al., 1992 PhoP and PhoQregulated genes (prg) Miller et al., 1989 Behlau and Miller, 1993; 1994Porins Tufano et al., 1988 Serum resistance peptide Hackett et al., 1987Virulence factors Abshir and Neidhardt, 1993 Loos and Wassenaar, 1994Mahan et al., 1995 Sansonetti, 1992 Virulence plasmid Gulig and Curtiss,1987 Rhen et al., 1993 Riikonen et al., 1992 spvB (virulence plasmid)Fierer et al., 1993 traT (virulence plasmid) Rhen and Sukupolvi, 1988ty2 Elsinghorst et al., 1989 Abshiro et al., 1993, J. Bacteriol.175:3734-3743 Alpuche-Aranda et al., 1992, Proc. Natl. Acad. Sci. USA89:10079-83 Alpuche-Aranda et al., 1994, J. Exp. Med. 179:601-6088Baumler et al., 1994, Infect. Immun. 62:1623-1630 Behlau et al., 1993,J. Bacteriol. 175:4475-4484 Belden et al., 1994, Infect. Immun.62:5095-5101 Betts et al., 1992, Can. J. Microbiol. 38:852-7 Boichenkoet al., 1987, Bull. Eksp. Biol. Med. 103:190-2 Boichenko et al., 1988,Zh. Mikrobiol. Epidemiol. Emmunobiol. 7:9-11 Boichenko et al., 1985, Zh.Mkrobiol. Epidemiol. Immunobiol. 12:67-9 Bowe et al., 1994, MethodsEnzymol. 236:509-26 Buchmeier et al., 1989, Infect. Immun. 57:1-7Buchmeier et al., 1990, Science 248:730-732 Buchmeier et al., 1995, J.Cldn. Invest. 95:1047-53 Buchmeier et al., 1993, Mol. Microbiol.7:933-936 Dragunsky et al., 1989, J. Biol. Stand. 17:353-60 Emoto etal., 1993, J. Immunol. 150:3411-3420 Ernst et al., 1990, Infect. Immun.58:2014-2016 Elsinghorst et al., 1989, Proc. Natl. Acad. Sci. USA86:5173-5177 Fields et al., 1986, Proc. Natl. Acad. Sci. USA 83:5189-93Fields et al.,1989, Science 243:1059-62 Fierer et al., 1993, Infect.Immun. 61:5231-5236 Gianella et al., 1973, J. Infect. Dis. 128:69-75Galan et al., 1989, Microb. Pathog. 6:433-443 Galan et al., 1990,Infect. Immun. 58:1879-1885 Ginocchio et al., 1992, Proc. Natl. Acad.Sci. USA 89:5976-5980 Gulig et al., 1987, Infect. Immun. 55:2891-901Hackett et al., 1987, J. Infect. Dis. 1 55:540-549 Heffeman et al.,1992, J. Bacteriol. 174:84-91 Ishibashi et al., 1992, Microb. Pathog.13:317-323 Libby et al., 1994, Proc. Natl. Acad. Sci. USA 91:489-493Loos et al., 1994, Immun. Infekt. 22:14-19 Mahan et al., 1995, Proc.Natl. Acad. Sci. USA 92:669-673 Miller et al., 1989, Proc. Natl. Acad.Sci. USA 86:5054-5058 Miller et al., 1992, Infect. Immun. 60:3763-3770Nunoshiba et al., 1993, Proc. Natl. Acad. Sci. USA 90:9993-9997 Pollacket al., 1986, Nature 322:834-836 Pulkkinen and Miller, 1991, J.Bacteriol. 173:86-93 Rhen et al., 1993, Mol. Microbiol. 10:45-56 Rhen etal., 1988, Microb. Pathog. 5:275-285 Riikonen et al., 1992, Microb.Pathog. 13:281-291 Sansonetti, 1992, Rev. Prat. 42:2263-2267 Stone etal., 1992, J. Bacteriol. 174:3945-3952 Tufano et al., 1988, Eur. J.Epidemiol. 4:110-114

Yet another method for the attenuation of the isolated vectors is tomodify substituents of the microorganism which are responsible for thetoxicity of that microorganism. For example, lipopolysaccharide (LPS) orendotoxin is primarily responsible for the pathological effects ofbacterial sepsis. The component of LPS which results in this response islipid A (LA). Elimination or mitigation of the toxic effects of LAresults in an attenuated bacteria since 1) the risk of septic shock inthe patient would be reduced and 2) higher levels of the bacterialvector could be tolerated. Rhodobacter (Rhodopseudomonas) sphaeroidesand Rhodobacter capsulatus each possess a monophosphoryl lipid A (MLA)which does not elicit a septic shock response in experimental animalsand, further, is an endotoxin antagonist. Loppnow et al., 1990, Infect.Immun. 58:3743-3750; Takayma et al., 1989, Infect. Immun. 57:1336-1338.

Known similarities in lipid metabolism and genetic organization of lipidmetabolic genes between Rhodobacter sphaeroides and other gram negativebacteria and the ability of Rhodobacter genes to complement E. colimutations (Benning and Somerville, 1992(A), J. Bacteriol. 174:6479-6487;1992(B), J. Bacteriol. 174:2352-2360; Carty et al., 1994, FEMSMicrobiol. Lett. 118(3):227-231) demonstrate that, for example,Salmonella and other bacteria can be genetically altered to produce MLA,thereby reducing its potential of inducing septic shock. A preferredembodiment of the present invention is a Salmonella spp. strain thatexpresses MLA rather than LA and also expresses HSV TK under the controlof a tumor-specific promoter.

As an illustrative example, the generation of MLA producing Escherichiacoli or Salmonella typhimurium entails constructing a DNA gene librarycomposed of 10 kB fragments from Rhodobacter sphaeroides which isgenerated in λgtll or pUC19 plasmids and transfected into E. coli.Clones which produce MLA are positively selected by using an antibodyscreening methodology to detect MLA, such as ELISA. In another exampleone generates a cosmid library composed of 40 kB DNA fragments fromRhodobacter sphaeroides in pSuperCos which is then transfected intoSalmonella typhimurium. Clones which produce MLA are positively selectedby using an antibody screening methodology to detect MLA, such as ELISA.

Yet another example for altering the LPS of Salmonella involves theintroduction of mutations in the LPS biosynthetic pathway. Severalenzymatic steps in LPS biosynthesis and the genetic loci controllingthem in both E.coli and Salmonella typhimurium have been identified(Raetz, 1993, J. Bacteriol. 175:5745-5753 and references therein).Several mutant strains of Salmonella typhimurium and E. coli have beenisolated with genetic and enzymatic lesions in the LPS pathway. One suchmutant, firA is a mutation within the gene that encodes the enzymeUDP-3-O(R-30 hydroxymyristoyl)-glycocyamine N-acyltransferase, thatregulates the third step in endotoxin biosynthesis (Kelley et al., 1993,J. Biol. Chem. 268:19866-19874). Salmonella typhimurium and E.colistrains bearing this type of mutation produce a lipid A that differsfrom wild type lipid A in that it contains a seventh fatty acid, ahexadecandic acid (Roy and Coleman, 1994, J. Bacteriol. 176:1639-1646).Roy and Coleman demonstrated that in addition to blocking the third stepin endotoxin biosynthesis, the firA⁻ mutation also decreases enzymaticactivity of lipid A 4′ kinase that regulates the sixth step of lipid Abiosynthesis.

Once the strain has been attenuated by any of the methods known in theart, the stability of the attenuated phenotype is important such thatthe strains do not revert to a more virulent phenotype during the courseof treatment of a patient. Such stability can be obtained, for example,by providing that the virulence gene is disrupted by deletion or othernon-reverting mutations on the chromosomal level rather thanepistatically or that the “suicide gene” is stably integrated into thebacterial chromosome.

Another method of insuring the attenuated phenotype is to engineer thebacteria such that it is attenuated in more than one manner, e.g., amutation in the pathway for lipid A production, such as the firA⁻mutation (Hirvas et al., 1991, EMBO J. 10:1017-1023) and one or moremutations to auxotrophy for one or more nutrients or metabolites, suchas uracil biosynthesis, purine biosynthesis, and arginine biosynthesisas described by Bochner, 1980, J. Bacteriol. 143:926-933. In a moreprefered embodiment of the invention, the bacterial vector whichselectively targets tumors and expresses a pro-drug converting enzyme isauxotrophic for uracil, aromatic amino acids, isoleucine and valine andsynthesizes an altered lipid A.

6.3. In Vitro Cancer Diagnostics and in Vivo Treatment of Solid TumorsUsing Isolated Vectors and Other Vectors 6.3.1. In Vitro Diagnostics

An embodiment of the present invention is to provide methods for use ofthe vectors of the present invention in in vitro diagnostic assays anddiagnostic kits for the detection of solid tumor cancers, including butnot limited to melanoma. Also, the kits may comprise tumor-specificnon-attenuated vectors. The In vitro diagnostic assays and kits arebased on the enhanced specificity towards a cancerous cell rather thanits non-cancerous counterpart cell of a vector. For example, and not byway of limitation, a putative solid tumor is biopsied from a patient.The tumor biopsy is minced and digested to a suspension of single cells.Aliquots of the suspension and a non-cancerous counterpart or controlcell are cultured and infected with a tumor-specific vector according tothe present invention.

After an incubation period, the number of tumor-specific microorganismswhich attached to and/or infected the biopsied cells as compared to thenon-cancerous counterpart or control cells is determined by any methodknown to those skilled in the art. A higher number of vectors foundassociated with the target cell as compared to the non-cancerouscounterpart or control cells indicates that the target cell iscancerous, for example, about 5-10 times as many vectors will infect atumor cell compared to a non-cancerous control cell. A non-cancerouscounterpart or control cell is the normal cell from which the tumor cellis derived, for example, for melanoma cells the non-cancerouscounterpart or control cell is melanocyte cells, for colon cancer thecounterpart cell is colon epithelial cells. In one embodiment the ratiois determined as the number of vectors/target cell. In anotherembodiment, after infection, the cells are fixed and treated with astain or antibody which recognizes DNA so that the vector DNA present inthe target cell cytoplasm is visualized. The presence of DNA in thetarget cell cytoplasm indicates that the biopsied target cells arecancerous. In one embodiment of the present invention the diagnosticmethod comprises exposing a sample of cells which are suspected of beingcancer cells to a tumor-specific vector or microorganism. The methodalso comprises exposing a sample of non-cancerous counterpart cells tothe tumor-specific vector or microorganism as a comparative control.After incubating for a time period in which the microorganisms canattach to and/or infect cancer cells, the infectivity of themicroorganism or vector for the cells suspected of being cancerous andthe non-cancerous counterpart control cells can be compared.

The diagnostic kits of the present invention comprise an effectiveamount of a tumor-specific vectors. The kits can further comprise anappropriate amount of non-cancerous control cells. The vector and/orcells may be supplied either frozen, lyophilized or growing on solid orin liquid medium. The diagnostic kits can further comprise inertingredients and other kit components such as vials, packaging componentsand the like, which are well known to those skilled in the art.

In certain embodiments, the vectors useful for the methods of diagnosisof the present invention can further comprise tumor-specific, attenuatedor non-attenuated vectors. In other embodiments, the kits of the presentinvention can comprise tumor-specific, attenuated or non-attenuatedvectors.

For illustrative examples of in vitro diagnostics of solid tumorcancers, including but not limited to melanoma, see Sections 22, 25 and26.

6.3.2. In Vivo Treatment of Solid Tumors

The vectors for use in in vivo cancer treatment are a subset of thevectors of the present invention. The vectors for in vivo treatment havebeen attenuated such that, when administered to a host, the vector hasbeen made less toxic to the host and easier to eradicate from the host'ssystem. In a preferred embodiment, the vectors are super-infective,attenuated and specific for a target tumor cell. In a more preferredembodiment, the vectors are also sensitive to a broad range ofantibiotics.

In addition, the isolated vectors can encode “suicide genes”, such aspro-drug converting enzymes or other genes, which are expressed andsecreted by the vector in or near the target tumor. The gene can beunder the control of either constitutive, inducible or cell-typespecific promoters. In a preferred embodiment, a suicide gene isexpressed and secreted only when a vector has invaded the cytoplasm ofthe target tumor cell, thereby limiting the effects due to expression ofthe suicide gene to the target site of the tumor.

In a preferred embodiment, the vector, administered to the host,expresses the HSV TK gene. Upon concurrent expression of the TK gene andadministration of ganciclovir to the host, the ganciclovir isphosphorylated in the periplasm of the microorganism which is freelypermeable to nucleotide triphosphates. The phosphorylated ganciclovir, atoxic false DNA precursor, readily passes out of the periplasm of themicroorganism and into the cytoplasm and nucleus of the host cell whereit incorporates into host cell DNA, thereby causing the death of thehost cell.

Another embodiment of the present invention is to provide methods oftreatment of solid tumor cancers with isolated attenuated vectors of thepresent invention. For example, a patient is diagnosed with a solidtumor cancer by any method known in the art, including the in vitrodiagnostic methods of the present invention. The vector used in thetreatment may already be isolated using the methods of the presentinvention with target cell lines or using model tumors in mice of thetarget tissue. In another embodiment, the biopsied tumor cells are usedin the selection assay for isolating a vector which is super-infectiveand tumor-specific for the tumor of the patient. In a preferredembodiment the vector is genetically modified, for example, to lackvirulence factors, express a suicide gene or both as described inSection 6.2.2. In addition, the isolated vector is analyzed forsensitivity to antibiotics to insure the eradication of the vector fromthe patient's body after successful treatment or if the patientexperiences complications due to the administration of the isolatedvector.

When administered to a patient, e.g., an animal for veterinary use or toa human for clinical use, the vectors can be used alone or may becombined with any physiological carrier such as water, an aqueoussolution, normal saline, or other physiologically acceptable excipient.In general, the dosage would range from about 1 to 1×10⁸ c.f.u./kg,preferably about 1 to 2×10² c.f.u./kg.

The vectors of the present invention can be administered by a number ofroutes, including but not limited to: orally, topically, injectionincluding, but limited to intravenously, intraperitoneally,subcutaneously, intramuscularly, intratumorally, i.e., direct injectioninto the tumor, etc.

The following series of examples are presented by way of illustrationand not by way of limitation on the scope of the invention.

7. EXAMPLE Isolation of Super-Infective, Tumor-Specific Salmonellatyphimurium in Vitro

7.1. Mutagenesis Before Isolation of Super-Infective, Tumor-SpecificClones

A culture of Salmonella typhimurium strain #14028 was grownexponentially at 37° C. in minimal medium 56 plus glycerol (0.5%) toOD⁶⁰⁰=0.3, then chilled on ice. An aliquot was removed so that theculture could be titered for colony forming units (c.f.u.) on LB agarplates. The culture was washed and resuspended in Na citrate (0.1M, pH5.5), incubated with fresh nitrosoguanidine (NG, 50 μg/ml, 20 minutes,37° C.), washed once by centrifugation, resuspended in medium 56,chilled, and again an aliquot was removed so that the culture could betitered for c.f.u. on LB agar plates. Another aliquot of the NG treatedbacteria was diluted (1:5) into LB broth and grown to stationary phasefor storage frozen at −80° C. in 12% glycerol.

The remaining bacteria were irradiated with ultraviolet light,dose=50J/m2, λ=254 nm). An aliquot was removed and the cells were thentitered for c.f.u. on LB agar plates, with another aliquot diluted 1:4into LB broth, grown to stationary phase, and stored frozen at −80° C.in 12% glycerol.

The mutagenesis procedures produced an increase in the number ofmutations in the strain by four criteria: 1) decreased survival of thebacteria following mutagenesis (nitrosoguanidine=6-fold; ultraviolet Birradiation=400-fold); 2) increased frequency of auxotrophic(nutritional requiring) mutants to (2%); 3) increased frequency ofmaltose mutants to (2%); 4) increased frequency of galactose⁻ mutants to(0.5%).

7.2. Isolation of Super-Infective Salmonella Typhimurium Clones #70 and#71 Specific For Cancer Cells

A population of Salmonella typhimurium wild type strain #14028 wasmutagenized as described in Section 7.1 with nitrosoguanidine and UVirradiation. Briefly, the bacteria were grown exponentially at 37° C. inminimal medium 56 plus glycerol to OD⁶⁰⁰=0.3, chilled on ice, washed,resuspended in Na citrate with 50 μg/ml nitrosoguanidine and incubatedfor 20 minutes at 37° C. The bacteria were washed once by centrifugationand resuspended in medium 56. The bacteria were then irradiated with UVlight at a dose of 50J/m², λ=254 nm.

Prior to infection by Salmonella, human M2 melanoma cells wereinoculated into Corning Tissue Culture flasks (25 cm²) at approximately2×10⁵ cells/flask in 4 ml DMEM cell culture medium containing penicillin(100 units/ml), and streptomycin (100 μ/ml), and incubated overnight ina 37° C., gassed (5%CO2), humidified incubator. The next day the cellswere rinsed twice with prewarmed Dulbecco's Minimal Essential mediumsupplemented with 10% fetal bovine serum (DMEM/FBS) and no antibiotics.

The mutated population of Salmonella typhimurium was cultured on LB agarovernight at 37° C. or in a liquid culture. The following day thebacteria were transferred with a platinum wire loop to LB broth or toDMEM/FBS, adjusted in concentration to OD⁶⁰⁰=0.1 (approximately 2×10⁸c.f.u./ml), and subjected to further growth at 37° C. on a rotator.Following growth to the desired population density (monitored at anoptical density of 600 nm) the bacteria were diluted to a concentrationof 10⁶ c.f.u./ml in DMEM/FBS, and incubated at 37° C. an additional 20minutes.

The mutagenized bacterial population was subjected to a single cycle ofinfection into- and isolation from human M2 melanoma cells in culture.Portions of the mutagenized population were grown clonally on agar and20 clones of Salmonella typhimurium were separately isolated and testedfor their individual infectivity toward human M2 melanoma cells. Thebacteria were added to animal cell cultures in 25 cm² Corning TissueCulture flasks at 4 ml/flask, and incubated with the animal cells in agassed (5%CO²/95% air), humidified incubator at 37° C. After a 15 minuteincubation with the animal cells the bacteria-containing medium waspoured off and the cultures were rinsed gently with warmed DMEM/FBS (4ml) containing gentamicin sulfate (20 μg/ml), an antibiotic that killsextracellular but not intracellular bacteria. The gentamicinsulfate-containing medium was poured off, fresh DMEM/FBS/gentamicinsulfate medium was added, and the cells were incubated for 60 minutes at37° C. Following the 60 minute incubation with gentamicin sulfate, themedium was poured off, the flasks were rinsed 1× with DMEM/FBS (withoutgentamicin sulfate), and 1 mM EDTA or an EDTA/trypsin solution (SigmaChemicals, 1×) in Ca⁺⁺/Mg⁺⁺ free physiological saline (4 ml) was added.After incubating with EDTA or EDTA/trypsin for 20 minutes at 37° C., theflasks were shaken to suspend the animal cells, and aliquots wereremoved for quantitation. Animal cells were quantitated in a CoulterCounter™ size-dependent particle counter (Coulter Electronics, Inc.) andbacteria were quantitated by plating aliquots on LB agar, incubating at37° C., and counting colonies. Quantitation was expressed as the numberof infecting (gentamicin resistant) bacteria/10⁶ animal cells.

Two clones, “70” and “71”, were found to be super-infective of melanomacells, with infection capacities 5-10-fold greater than the mutagenizedwild type strain (data not shown). Clones 70 and 71 were also assessedfor their relative specificity of the following human cells in culture:M2 melanoma cells and normal human melanocytes; “CaCo” colon cancercells and normal human colon epithelium #1790 as depicted in Table 4(A).

TABLE 4(A) SPECIFIC INVASION OF S. TYPHIMURIUM INTO MELANOMA VSMELANOCYTES AND COLON CANCER VS COLON EPITHELIUM IN CELL CULTURE: CLONES“70” AND “71”⁺ Human Infecting Salmonella/10⁶ human cells: Cell LineClone 70 (ratio)* Clone 71 (ratio)* normal 1.4 ± 0.2 33 10⁶ 1.2 ± 0.3 ×10⁶ melanocytes M2 melanoma 7.3 ± 2.0 × 10⁶ (5.2) 5.7 ± 0.7 × 10⁶ (4.8)colon 1.5 ± 0.1 × 10⁶ 0.8 ± 0.2 × 10⁶ epithelium (#1790) colon 7.2 ± 2.0× 10⁶ (4.8) 2.3 ± 0.3 × 10⁶ (2.9) carcinoma (CaCo) *cancer cell:normalcounterpart cell ⁺Results represent averages ± SD for triplicateinfections.

The bacterial clones #70 and #71 showed strong invasion preference formelanoma and colon cancer cells over that for normal melanocytes andnormal colon epithelial cells.

7.3. Isolation of Salmonella Typhimurium Super-Infective Clone #72 ByCycling in Vitro Cell Culture

Salmonella wild type strain #14028 was mutagenized with nitrosoguanidineand ultraviolet B irradiation as described in Section 7.1. A startingpopulation of 5×10⁸ mutagenized bacteria was grown to OD⁶⁰⁰=0.450,diluted in DMEM/FBS to a concentration of 5×10⁷ c.f.u./ml, and allowedto infect human M2 melanoma cells for 15 minutes. Infecting bacteriawere isolated from the melanoma cells, and again allowed to infectfresh, uninfected populations of melanoma cells. The 2^(nd) round ofinfecting bacteria were again isolated and subjected to further cyclesof infection into, and isolation from, human M2 melanoma cells. Afterthe completion of 4 such cycles, the population of melanoma-cycledbacteria which is designated 14028^(pop-1) was then plated on agar and100 individual clones were picked and tested for their ability, comparedto wild type bacteria, to infect M2 melanoma cells. The results of theselection process on 14028^(pop-1) and selected population sub-clonesare detailed in Table 5.

Additionally, an aliquot of 14028^(pop-1) was subjected to two furthercyclings in M2 melanoma cells. This 6×-cycled population was thensubjected to 7 cycles of negative selection against normal humanmelanocytes. The 6×-cycled population was added to a culture of normalhuman melanocytes and incubated for 15 minutes. The supernatant wascollected and was then added back to a fresh culture of normal humanmelanocytes. This negative selection procedure was carried out 7 times.This 6×-7× cycled population was again added to M2 melanoma cells,allowed to infect the melanoma cells for 15 minutes, and the bacteriawere then collected from the M2 cells. This 6×-7×-1× cycled populationwas designated 14028^(pop-2).

The mixed population of 4 times cycled Salmonella typhimurium,designated 14028^(pop-1), showed a 3-fold increased infectivity ofmelanoma cells over that of the starting mutagenized population of wildtype bacteria. Of the 100 clones isolated from population 14028^(pop-1)of Salmonella, two clones, #6 and #72, were found to be significantlysuper-infective of melanoma cells. The remaining bacterial clones showedinfectivity that was similar to or below that of the wild type strain.In the experiment presented in Table 5, clone #6 was about 25-fold, andclone #72 was about 55-fold more infective than the mutagenized wildtype strain during a 15 minute infection period. Escherichia coli,strain K-12, #CSH 101, was at least two orders of magnitude lessinfective than wild type Salmonella typhimurium, thus, demonstrating thenatural ability of S. typhimurium to infect certain animal cells.

TABLE 5 INFECTION OF M2 HUMAN MELANOMA CELLS WITH VARIOUS ISOLATEDSALMONELLA TYPHIMURIUM POPULATIONS IN CULTURE⁺ melanoma cells SalmonellaStrain Infecting Bacterial/10⁶ (% wild type) Wild type S. typhimurium3.8 ± 3.0 × 10⁴ 100 #14028 (mutagenized) #14028^(pop-1) 1.1 ± 0.4 × 10⁵290 Clone #6 8.6 ± 1.0 × 10⁶ 2260 Clone #72 2.1 ± 0.2 × 10⁶ 5500 E. coliK-12 <10² <1 ⁺Results represent averages ± SD for triplicate infections.

Over several such experiments shown in Table 5, the infectivity of clone#72 toward melanoma cells varied from 5- to 90-fold over that of thewild type strain. This variation seemed to depend on the bacterialgrowth density prior to infection of melanoma cells. Therefore, theeffect of population density on relative infectivity between wild typeand clone #72 was determined.

Wild type S. typhimurium and super-infective clone #72 were grown as alawn on LB agar plates. Portions of the cultures were removed with aplatinum loop and inoculated into LB broth at a concentration ofapproximately 2×10⁸ c.f.u./ml (OD⁶⁰⁰=0.1). The cultures were then placedon a rotator at 37° C. and optical densities were monitored as afunction of population density. At the optical densities indicated,aliquots of bacteria were removed, diluted in melanoma growth medium(DMEM/10% FBS) to a density of 1×10⁶ c.f.u./ml, and allowed to infecthuman M2 melanoma cells. Infectivity assays were carried out asdescribed. The results are shown in Table 6.

TABLE 6 INFECTIVITY OF WILD TYPE S. TYPHIMURIUM AND SUPER-INFECTIVECLONE #72 TOWARDS HUMAN MELANOMA CELLS: EFFECT OF BACTERIAL POPULATIONDENSITY⁺ Optical Density: Salmonella/10⁶ melanoma Infectivity ratio:(600 nm) Clone #72 Wild type (Clone 72:wild type) 0.200 -0- -0- (noinfectivity) 0.300 9.0 × 10³ -0- (infinite) 0.400 5.0 × 10⁴ -0-(infinite) 0.500 4.5 × 10⁵ 5.0 × 10³ 90:1 0.600 1.2 × 10⁶ 3.7 × 10⁴ 32:10.700 2.3 × 10⁶ 2.5 × 10⁵  9:1 0.800 3.2 × 10⁶ 5.6 × 10⁵  6:1 0.900 3.6× 10⁶ 7.3 × 10⁵  5:1 ⁺Results represent averages of duplicateexperiments. Variations between duplicates were < ± 15%.

The results demonstrate that infectivity of both bacterial strains washighly dependent on bacterial population density prior to infection,however, clone #72 was proportionately more infective than the wild typestrain at low population densities.

The results shown in Tables 5 and 6 were also confirmed by phase andlight microscopy which revealed super-infectivity of a 10×melanoma-cycled population of Salmonella typhimurium designated “M10” asshown in FIGS. 2 and 3. It was also found that wild type strain 14028and clone 72 infected human M2 melanoma cells equally well when grownunder anaerobic conditions prior to infection. However, when the strainsare grown under aerobic conditions, strain 14028 was strongly suppressedin infectivity, whereas clone 72 remained induced. Thus, clone 72 wasinfective under either anaerobic or aerobic growth conditions andsuperinfective compared to wild type under aerobic growth conditions.

7.4. Preferential Selectivety of S. Typhimurium For Cancer Cells: WildType Strain vs. Super-Infective Clone #72

Super-infective Salmonella typhimurium clone #72 isolated in Section 7.3was compared to the non-mutagenized wild type strain #14028 for relativeinfectivity of human M2 melanoma cells, normal human melanocytes, coloncancer cells and normal colon epithelium. The results are shown in Table7.

TABLE 7 TUMOR SPECIFICITY OF WILD TYPE S. TYPHIMURIUM ANDSUPER-INFECTIVE CLONE #72 TOWARD VARIOUS NORMAL AND CANCEROUS CELLS INCULTURE⁺ Animal Infecting Salmonella/10⁶ animal cells Cell Line wildtype (ratio)* Clone #72 (ratio)* normal 1.2 ± 0.7 × 10⁴ 2.7 ± 0.4 × 10⁵melanocyte (foreskin, human) M2 human 2.5 ± 0.6 × 10⁴ (2.1) 1.7 ± 1.1 ×10⁶ (6.3) melanoma normal colon 6.6 × 0.8 × 10³ 5.2 ± 3.0 × 10⁵epithelium (1790, human) colon cancer 3.0 ± 2.0 × 10⁴ (4.6) 9.5 ± 3.0 ×10⁵ (1.8) (HTB 39, human) “normal” 1.8 ± 1.5 × 10⁴ 5.5 ± 1.4 × 10⁵fibroblast (3T3, mouse) transformed 2.4 ± 0.6 × 10⁴ (1.3) 4.6 ± 0.8 ×10⁶ (8.4) macrophage (J774, mouse) ⁺Results represent averages ± SD fortriplicate infections. *cancer cell:normal counterpart cell

Each of the two bacterial strains showed invasion preference for humancancer cells over normal cells. Clone #72 was super-infective in allcases when compared to the wild type strain. Further, clone #72 showed asignificantly higher degree of invasion specificity for human melanomacells over normal melanocytes than the wild type strain did.

7.5. Infectivity of Salmonella typhimurium Wild Type Strain 14028 andSuperinfective Clone 72 Toward Various Human Carcinomas in Culture

In another series of experiments, the relative infectivities of clone 72and wild type strain 14028, toward a variety of human carcinomas growingin culture, was determined. The experimental protocol used is describedin Section 7.2. Results are presented in Table 7(A).

TABLE 7(A) INFECTIVITY OF SALMONELLA TYPHIMURIUM WILD TYPE ANDSUPERINFECTIVE CLONE 72 TOWARD VARIOUS HUMAN CARCINOMAS IN CULTURESalmonella/10⁶ Human Cells Ratio Origin of Wildtype: Clone # 72:140 CellLine Primary Tumor 14028 72 28 M2 melanoma 4.0 ± 3.8 × 10⁴ 4.2 ± 3.5 ×10⁵ 11:1  HTB57 lung 2.8 ± 1.3 × 10³ 4.5 ± 2.1 × 10⁴ 16:1  HTB183 lung1.0 ± 0.3 × 10⁵ 4.1 ± 1.8 × 10⁵ 4:1 HTB54 lung 2.1 ± 0.7 × 10⁴ 1.7 ± 0.2× 10⁵ 8:1 A549 lung 3.7 ± 5.6 × 10⁴ 4.5 ± 4.9 × 10⁵ 12:1  CRL1740prostate 2.3 ± 0.4 × 10⁵ 1.8 ± 0.2 × 10⁵ 8:1 CRL1611 kidney 3.2 ± 1.2 ×10⁵ 1.8 ± 0.3 × 10⁵ 6:1 HTB52 liver 1.8 ± 0.3 × 10⁵ 2.6 ± 0.8 × 10⁵1.4:1   MCF7 breast 7.3 ± 2.6 × 10⁴ 3.6 ± 0.9 × 10⁵ 5:1 Resultsrepresent averages ± SD for n = 3-9 separate infections.

Both the wild type strain 14028 and clone #72 were able to infect eachof the human cancer cells tested in culture. In all cases, clone 72 wassuperinfective compared to the wild type strain.

However, human lung line HTB57 was significantly less receptive toSalmonella typhimurium infectivity when compared to other cancer celllines tested. In yet another series of experiments, the human lung lineHTB57 was implanted into mice. In 10 of 10 nu/nu mice implanted with1×10⁷ HTB57 cells, no tumor “takes” were observed, even after severalmonths. Whether or not these cells were receptive to Salmonellainfection when grown as tumors was not determined.

7.6. Discussion

In summary, the results demonstrate the following: a) infectivity of S.typhimurium is dependent upon population density of the bacteria and b)super-infective clone #72 differs from the wild type strain in itsincreased infectivity of melanoma cells at all bacterial populationdensities and especially at low population densities under aerobicgrowth conditions. The ability to infect at low bacterial populationdensities is an advantage in the use of clone #72 as a tumor-specificvector, since it would allow for a lower c.f.u. of bacteria inoculatedinto the cancer patient, thus reducing the risk of septic shock in thepatient. Additionally, the results demonstrate methods for the isolationof super-infective, tumor-specific mutants of S. typhimurium. Suchmutants are represented by clones #6, #70, #71 and #72 that wereisolated via enrichment procedures for melanoma infectivity by thebacteria. The results further demonstrate that wild type S. typhimuriumexhibits specificity for human cancer cells over normal human cells inculture. Further, although clone 72 was originally selected forsuperinfectivity toward human melanoma cell line M2, it was additionallyfound to be superinfective toward human colon cancer cells andtransformed mouse macrophages, when compared to the wild type strain14028 (see Table 7). The expression of super-infectivity andtumor-specificity of isolated mutant clones represent attenuation of thebacteria and present distinct advantages for the use of such Salmonellaclones as tumor-specific vectors in the diagnosis and therapy of humancancer.

8. EXAMPLE Selection For Salmonella typhimurium Mutants With ChemotacticAbilities Toward Melanoma Secretory Products in Vitro

The melanoma cells were an artificially-produced hybrid line isolatedfrom a polyethylene glycol induced fusion between Cloudman S91 mousemelanoma cells and peritoneal macrophages from a DBA/2J mouse. Thehybrid cell line used herein was termed Cloudman S91 melanoma/macrophagehybrid #48. The hybrid cell line formed rapidly growing metastasizingtumors in DBA/2J mice, Pawelek et al., 1995, J. Invest. Dermatol.104:605. 5×10⁶ Cloudman S91 melanoma/macrophage hybrid #48 cells werecultured at 30° C. in a gassed, humidified incubator in 75cm² cultureflasks in DMEM/FBS culture medium containing 10% fetal bovine serum andno antibiotics. Control flasks containing DMEM/FBS but no melanoma cellswere incubated in parallel. After 72 hours, the media were removed,aseptically filtered through 0.45μ filters, and stored at 4° C.

Salmonella typhimurium super-infective clone #72 described above wassubjected to mutagenesis with nitrosoguanidine and UV. The mutagenesisprocedures produced an increase in the number of mutations in Clone #72similar to that shown earlier when the wild type strain #14028 wasmutagenized. This mutagenized population of clone 72 (“72^(mut)”) wasfurther used to select for mutants with enhanced chemotactic abilitiestoward melanoma cell secretory products, i.e., melanoma-conditionedculture media.

Procedures for loading capillary tubes with potential chemotacticattractants were modified from Adler (Adler, 1973, J. GeneralMicrobiology 74:77-91). Control and melanoma-conditioned culture mediadescribed above, were loaded into 2λ capillary tubes (MICROCAPS™,capillary tubes, Drummond Scientific Co.) as described by Adler. Thecapillaries were handled with forceps. One end was sealed in a flame;the capillary was then quickly passed several timers through the flameand immediately plunged open end down into a 10 ml beaker containing 1ml control or melanoma conditioned culture medium. As the capillarycooled (about 10 minutes), liquid was drawn in about 1 cm.

Salmonella typhimurium, growing at 37° C. in LB were collected bycentrifugation and resuspended in control DMEM/FBS culture mediumcontaining a concentration of 10⁸ c.f.u./ml. Aliquots (200 μl, 2×10⁷c.f.u.) were pipetted into 1.5 ml microfuge tubes. Loaded capillarytubes (described above) were then inserted open end down into theBeckman microfuge tubes containing the Salmonella typhimurium, and theassay was begun by incubating at 37° C. After 30 to 60 minutes, thecapillary tubes were removed with forceps, the sealed ends were brokenoff with wire cutters, and the capillaries were transferred to 15 mlconical centrifuge tubes containing 3 ml LB broth. It was important thatthe upper tips of capillary tubes were covered with LB broth in order toassure quantitative recovery of the bacteria via the centrifugation stepdescribed as follows. The capillaries within the centrifuge tubes werethen centrifuged (1000×g for 4 minutes) to force the bacteria out of thecapillaries. The bacteria were resuspended by vortexing, and aliquotswere spread onto LB agar plates for quantitation, Significant increasesin the number of bacteria entering the capillaries containingmelanoma-conditioned media compared to control-conditioned mediaindicated a chemotactic response of the bacteria to melanoma-secretedproducts.

Aliquots of mutagenized super-infective Salmonella typhimurium,“72^(mut)”, described above were placed on a rotor at 37° C., grown toan optical density of 0.4-0.6 at a wavelength of 600 nm, and subjectedto the chemotaxis procedures described above. The chemotaxis cyclingprocedure was repeated 4 times through successive challenges withmelanoma-conditioned culture medium. The population obtained after 4cycles was designated #72^(pop-2). After the 4th cycling, aliquots ofthe mixed populations of bacteria were frozen in glycerol. Additionalaliquots of the mixed population of Salmonella typhimurium obtained fromthe fourth cycling were then compared to an uncycled mixed population ofmutagenized clone 72 (“72^(mut)”) for relative chemotactic abilitiestoward control and melanoma-conditioned culture medium. The results areshown in Table 8.

TABLE 8 EVIDENCE FOR POSITIVE CHEMOTACTIC RESPONSES OF S. TYPHIMURIUM TOCONDITIONED GROWTH MEDIUM OF CULTURED MELANOMA CELLS⁺ Bacteria/CapillaryTube: Salmonella Strain Control Medium Conditioned Medium Ratio:#72^(mut) 1.2 × 10³ ± 0.2 4.4 × 10³ ± 2.7 3.7:1 (mutagenized, nocycling) #72^(pop-2) 0.5 × 10³ ± 0.1 1.8 × 10³ ± 0.4 3.6:1 (mutagenized,cycled cycled 4×) ⁺Results represent average ± S.D for quadruplicatecapillary tubes.

Both populations of bacteria tested showed positive chemotacticresponses to melanoma-conditioned culture medium overcontrol-conditioned medium, displaying an approximate 4:1 preference forthe melanoma-conditioned medium. Although the chemotactic response ofpopulation #72^(pop-2) was not statistically significant as compared tothe chemotactic response of population #72^(mut) for melanomaconditioned medium, the chemotactic response of population #72^(pop-2)was significantly reduced as compared to the chemotactic response ofpopulation #72^(mut) for control medium. Thus, the propensity ofpopulation #72^(pop-2) to enter capillary tubes containing controlmedium was significantly reduced. These results suggest that population#72^(pop-2) is less efficient in motility generally, however, uponexposure to melanoma-conditioned medium, population #72^(pop-2) showed achemotactic response equivalent to the control population.

Whatever the mechanisms for the different chemotactic phenotypesexpressed by the #72^(mut) and #72^(pop-2) populations of bacteria inTable 8, the results demonstrate that the phenotypes can be altered viathe selection procedure of exposing bacteria to successive challenges ofmelanoma-conditioned media. It is likely that the mixed populations ofmutagenized, chemotactically cycled bacteria isolated in theseexperiments contain a number of diverse mutants expressing likewisediverse phenotypes for the chemotactic response to melanomacell-conditioned medium.

9. EXAMPLE Isolation of Tumor-Specific Mutants of Salmonella typhimuriumBy Cycling in Vivo in Tumor-Bearing Mice

Tumor cells inoculated into DBA/2J mice from Cloudman S91melanoma/macrophage hybrid cell line #48 were used as the target tumorfor the selection of attenuated, tumor-specific Salmonella typhimurium.Super-infective Salmonella typhimurium clone #72 was mutagenized withnitrosoguanidine and UVB as described in Section 7.1 producing amutagenized population derived from clone #72. The mutagenesisprocedures produced an increase in the number of mutations in clone #72similar to that shown earlier when the wild type strain #14028 wasmutagenized. Cloudman melanoma/macrophage hybrid #48 cells wereinoculated (s.c.) into DBA/2J mice at a concentration of 10⁶ cells in0.1 ml saline/inoculated site and a total of 4 sites/mouse in theshoulder and flank regions. After 8-10 days, palpable tumors developed,and the mice were inoculated (i.p.) with the mutagenized Salmonellapopulation derived from super-infective clone #72. After 2 hours ofinfection, the mice were sacrificed, the tumors removed, weighed, andhomogenized in a teflon homogenizer in 5 vol (vol/wt) LB broth. Analiquot of the homogenate was then diluted about 1:4 in LB broth, placedon a rotator at 37° C., and incubated through 1-2 population doublings,should be monitored at OD⁶⁰⁰, in order to ensure the recovery of viablebacteria for successive inoculations into tumor-bearing mice. Theprocedure was repeated through 4 cycles of infection into mice, followedby recovery from tumors. At the beginning of each cycle, the number ofbacteria inoculated and the time of infection was reduced from theprevious cycle in order to increase the stringency of selection fortumor-specific mutants. The resultant population recovered after 4cycles was designated #72^(pop-1). The results of this procedure aredetailed in Table 9 below.

TABLE 9 SELECTION FOR MELANOMA-SPECIFIC SALMONELLA TYPHIMURIUM INTUMOR-BEARING MICE Infection Total # Bacteria Infection Total # BacteriaCycle Inoculated/mouse Time Recovered in Tumors* 1 1 × 10¹⁰ 120 min  2.1× 10⁷ 2 1 × 10⁹ 80 min 1.6 × 10⁶ 3 6 × 10⁸ 60 min 1.7 × 10⁶ 4 2 × 10⁶ 40min 1.4 × 10⁵ *Infecting Salmonella were pooled from 4-8 separate tumorsfor each cycle

These results demonstrate that infecting bacteria can be recovered fromtumors in vivo. These results also demonstrate that in vivo cyclingresults in an enriched population since fewer bacteria were isolatedthan were inoculated.

10. EXAMPLE Proliferation of Salmonella typhimurium Within MelanomaCells

Proliferation of a gene-delivering vector within target tissue can bothamplify the gene within the target tissue as well as allow one to reducethe titer of inoculated vector, thus reducing the risk of septic shockin the host. The following examples demonstrate that Salmonellatyphimurium proliferates in melanoma cells.

Proliferation Within Cultured Human M2 Melanoma Cells

It was found that Salmonella typhimurium proliferated within human M2melanoma cells in culture with doubling times of about 30 to 60 minutesas illustrated below. Wild type Salmonella strain #14028 andsuper-infective clone #72 were separately introduced into the culturemedia of human M2 melanoma cells 2×10⁵ melanoma cells/25 cm² tissueculture flask at 10⁶ bacterial c.f.u./ml culture medium. After 1 hour,gentamicin (20 μg/ml) was added to kill external, but not internalizedbacteria, and melanoma cells were harvested and assayed for the numberof internalized bacteria at the time points indicated. The results arepresented in Table 10.

TABLE 10 PROLIFERATION OF SALMONELLA TYPHIMURIUM WILD TYPE STRAIN #14028AND CLONE #72 WITHIN CULTURED HUMAN M2 MELANOMA CELLS⁺ Salmonella/Salmonella Strain Time (h) 10⁶ Melanoma Cells Fold Increase #14028 wildtype 1 6.8 × 10⁵ — 2 1.8 × 10⁶ 2.6× 4 1.8 × 10⁷  26× 6 5.4 × 10⁷  79×Clone #72 1 5.8 × 10⁶ — 2 8.0 × 10⁶ 1.4× 4 3.2 × 10⁷ 5.5× 6 1.4 × 10⁸ 24× ⁺The numbers represent averages for duplicate and triplicatedeterminations, with the variation between replicates < ± 25%.

10.2. Proliferation Within Melanoma Tumors Grown in Mice

DBA/2J mice were inoculated s.c. in four areas (left and right shouldersand flanks) with 10⁶ Cloudman S91 melanoma/macrophage hybrid #48 cells.After the appearance of palpable tumors (8-10 days) the mice werefurther inoculated (i.p.) with 2×10⁸ Salmonella typhimurium. TheSalmonella strains tested were wild type #14028 and super-infectiveclone #72. At 4 hours and 21 hours post-inoculation with bacteria, micewere bled orbitally, and then euthanized by anesthesia with metofane.Tumors and livers were removed aseptically, rinsed with sterile NaCl(0.9%), weighed, and homogenized in LB broth at a ratio of 5:1(vol:tumor wt). Bacteria were quantitated by plating the homogenatesonto LB plates, incubating overnight at 37° C., and counting bacterialcolonies. Numbers represent averages± S.D. The results for the 4 hourand 21 hour incubations of the bacteria in mice are detailed in Tables11(A) and 11(B).

TABLE 11 A. DISTRIBUTION OF SALMONELLA TYPHIMURIUM 4 HOURS FOLLOWINGINOCULATION (I.P.) INTO CLOUDMAN S91 MELANOMA-BEARING DBA/2J MICESalmonella/ Salmonella/ Salmonella Salmonella/ gm tumor gm liver Tumor/Strain ml Blood (wet wt) (wet wt) Liver wild type 6 × 10⁶ 8.9 ± 2.5 ×10⁴ 3.6 × 10⁶ 1:4 (n = 4) clone 72 2 × 10⁶ 3.5 ± 3.3 × 10⁴ 2.4 × 10⁵ 1:7(n = 4) B. DISTRIBUTION OF SALMONELLA TYPHIMURIUM 21 HOURS FOLLOWINGINOCULATION (l.P.) INTO CLOUDMAN S91 MELANOMA-BEARING DBA/2J MICESalmonella/ Salmonella/ Salmonella Salmonella/ gm tumor gm liver Tumor/Strain ml Blood (wet wt) (wet wt) Liver wild type 1.0 × 10⁴ 1.3 ± 0.8 ×10⁹ 4.4 × 10⁶  300:1 (n = 4) clone 72 6.7 × 10³ 2.1 ± 2.7 × 10⁹ 5.2 ×10⁵ 4000:1 (n = 4)

At 4 hours post-inoculation of Salmonella, there were fewer bacteria inthe tumors than in the blood stream and the liver for both wild typeclone 14028 and clone 72. However, by 21 hours, Salmonella were found ingreat abundance in the tumors so that the ratio of bacteria/g tissue intumors was 4,000:1 over that in the liver for super-infective mutantclone 72. After 21 hours post-inoculation of bacteria, the number ofSalmonella in the tumors was similar for both the wild type Salmonellastrain and clone 72, and was far greater than the total number ofSalmonella originally inoculated, indicating that both wild-type andclone 72 strains of bacteria proliferated within the tumors. Thus, theability of Salmonella typhimurium to infect melanoma cells andproliferate within them was expressed both in cell culture as seen inTable 10 and in tumors growing in mice as seen in Tables 11A and 11B.

The wild-type strain 14028 showed higher infectivity in liver than didclone 72. The higher infectivity of liver by the wild-type Salmonellawas consistent with the observed greater lethality of the wild typestain toward DEA/2J mice than that produced by clone 72 at highbacterial inocula (>10⁹ c.f.u./mouse, data not shown). Similar resultswere observed with C57BL\6J mice bearing B16F10 melanomas as seen inTable 18, Section 15.2. Together, these results demonstrate thatselection for strains of bacteria or other parasites with enhanced tumorspecificity in vitro yields mutant strains with attenuated host toxicityin vivo.

10.3. Distribution of Salmonella Typhimurium in Tumor-Bearing Mice

The following experiments demonstrate that Salmonella can localize toand proliferate within a tumor of an animal bearing eithermultiply-implanted subcutaneous melanoma tumors or naturally occurringmetastases.

10.3.1. Distribution of Salmonella Following Direct Inoculation IntoCloudman S91 Melanoma Tumors

DBA/2J mice were inoculated s.c. in four areas (left and right shouldersand flanks) with 10⁶ Cloudman S91 melanoma/macrophage hybrid site.Palpable tumors appeared 8-10 days post-inoculation, representativeanimals were selected, and 2 of the 4 tumors (right shoulder and leftflank) were directly inoculated with Salmonella typhimuriumsuper-infective clone #72 at c.f.u.'s of 7×10⁴ or 7×10⁶ bacteria/tumor.At 21 hours post-inoculation, mice were euthanized with metofane. Tumorsand livers were removed aseptically, rinsed with sterile NaCl (0.9%),weighed, and homogenized with NaCl at a ratio of 5:1 (vol:tumor wt).Bacteria were quantitated by plating the homogenates onto LB plates,incubating overnight at 37° C., and counting bacterial colonies. Theresults are shown in Table 12.

TABLE 12 DISTRIBUTION OF SALMONELLA TYPHIMURIUM CLONE 72 IN CLOUDMAN S91MELANOMA-BEARING MICE 21 HOURS FOLLOWING DIRECT INOCULATIONS INTO TUMORSInoculum/ Salmonella/ Salmonella/ tumor g tumor (wet wt) g liver (wetwt) Tumor/Liver 7.2 × 10⁴ Tumor 1* 1 × 10⁹ 3.0 × 10⁵ 3,300:1   Tumor 2 3× 10⁷ 100:1 Tumor 3 1 × 10⁸ 330:1 7.2 × 10⁶ Tumor 1* 4 × 10⁹ 5.0 × 10⁶800:1 Tumor 2 3 × 10⁹ 600:1 Tumor 3 3 × 10⁷  6:1 *inoculated tumor

In summary, two days post-inoculation of super-infective Salmonellatyphimurium clone #72 directly into specificized tumors, the Salmonellacould be found in distal, non-inoculated tumors. The amounts ofSalmonella found in the tumors far exceeded the amounts of Salmonellainoculated into the mice, proving that the Salmonella proliferatedwithin the tumors. The results demonstrate that Salmonella typhimuriumcan proliferate within a tumor, exit that tumor via the circulatorysystem, travel to a distant tumor, and proliferate within that distanttumor.

10.3.2. Distribution of Salmonella Into Cloudman S91 Melanoma Metastases

This experiment shows that the bacteria should be able to targetnaturally-occurring metastases of solid tumors.

1×10⁵ Cloudman S91 melanoma cells were inoculated s.c. in the tail of aDBA/2J mouse. After approximately four weeks, a soft tissue metastasis(˜0.5 g) developed with no visible evidence of a primary tumor in thetail. S. typhimurium clone 72 at 2×10⁵ c.f.u. was inoculated i.p. Themouse was sacrificed 48 hours post-inoculation, and the liver and tumorwere removed, homogenized in Luria broth, and quantitated for S.typhimurium by serial dilutions on LB agar plates.

The results shown in Table 12(A) demonstrate that Salmonella typhimuriumclone 72 can target and proliferate within a metastatic tumor.

TABLE 12(A) DISTRIBUTION OF SALMONELLA TYPHIMURIUM CLONE 72 IN A DBA/SJMOUSE WITH A SOFT-TISSUE MELANOMA METASTASIS* Tissue Salmonella/g tissueTumor/Liver Liver 3.1 × 10⁶ — Tumor 3.2 × 10⁹ 1000:1 *Results representdeterminations from a single animal.

11. EXAMPLE Antibiotic Sensitivity of Wild Type Salmonella TyphimuriumStrain 14028 and Super-Infective Mutant Clone 72

11.1. Sensitivity Tested in Vitro

Wild type Salmonella typhimurium strain 14028 and super-infective mutantclone 72 were tested for antibiotic susceptibility and were each foundto be sensitive to 12 different antibiotics currently used in treatingbacterial infections. The bacteria were tested according to the standardprotocol to determine antibiotic sensitivity as seen in clinicallaboratories, so that a patient is not given an antibiotic to which themicroorganism is resistant. The bacteria were tested for antibioticsusceptibility by subjecting them to the DISC DIFFUSION SUSCEPTIBILITYTECHNIQUE KIT™ antibiotic sensitivity assay kit (Remel Corp., Lenexa,Ks.). The data are presented in Table 13.

TABLE 13 ANTIBIOTIC SENSITIVITY Wild Type: Clone 72: Ampicillin S SCefoperazone S S Ceftazidime S S Cefuroxime S S Gentamicin S SMezlocillin S S Cefazolin S S Ciprofloxacin S S Unasyn S S Ceftriaxone SS TMP/SMX S S

11.2. Sensitivity Tested in Vivo

Susceptibility of Salmonella typhimurium cloned 72 to antibiotics wasfurther tested by injecting mice i.p. with bacteria, treating half ofthe mice with the antibiotic enrofloxacin, and observing the effects ofenrofloxacin, an active analog of ciprofloxacin, on the survival of themice.

Six week old C57B6 female mice were inoculated i.p. with 10⁵ cfuSalmonella typhimurium clone 72. After four days following inoculationwith bacteria, three of the mice were further inoculated i.p. with 100μg/0.1 ml BAYTRIL™ (enrofloxacin), and their drinking water wassupplemented with 25 μg/ml BAYTRIL™. After 4 days, all the mice weregiven fresh drinking water without BAYTRIL™. After a total of 21 days,all surviving mice were euthanized and the experiment was terminated.The results are shown below in Table 13(A).

TABLE 13(A) SURVIVAL OF C57B6 MICE INJECTED WITH SALMONELLA TYPHIMURIUMCLONE 72 ± BAYTRIL ® (enrofloxacin) IN DRINKING H₂O Conditions Avg. timeof Death ± S.D. no antibiotic 9.3 ± 5 days enrofloxacin (days 4-10post-inoculum) >21 days

Mice receiving bacteria only and no antibiotic died after an average of9 days following inoculation. Mice receiving bacteria followed byantibiotic treatment survived at least 21 days and showed no symptoms ofSalmonella toxicity when the experiment was terminated. Thus, theresults clearly demonstrate that mice can be rescued fromSalmonella-mediated death by treatment with the antibiotic enrofloxacin.These results are consistent with those presented in Table 13demonstrating antibiotic sensitivity of Salmonella typhimurium strains14028 and clone 72 by the DISC DIFFUSION SUSCEPTIBILITY TECHNIQUE KIT™antibiotic sensitivity assay kit.

The results further underscore the advantage of usingantibiotic-sensitive bacteria as vectors in human tumor therapy, sincethe bacteria can be eliminated by introduction of antibiotics whendesired.

12. EXAMPLE Enhanced Expression of Bacterial Promoters in Melanoma Cells

In a preferred embodiment of the present invention an isolatedsuper-infective vector, such as Salmonella typhimurium clone 72⁵⁻³⁻²which carries the HSV TK gene, the gene is specifically induced incancerous target cells as opposed to normal cells in the host body. Ithas been shown that there is a higher relative induction of severalSalmonella promoter genes, including pagB and pagC, (Miller et al.,1989, Proc. Natl. Acad. Sci. USA 86:5054-5058; Miller et al., 1992,Infect. Immun. 60:3763-3770; Alpuche Aranda et al., 1992, Proc. Natl.Acad. Sci. USA 89:10079-10083) when the bacteria invade macrophages asopposed to epithelial cells. In order to test whether these promotersare also activated when Salmonella invade melanoma cells, we usedSalmonella-bearing promoter constructs fused to the β-galactosidasereporter gene.

Human melanoma M2 cells, (Cunningham et al, 1992, Science, 255:325-327)human epithelial 1790 cells and mouse macrophage cell line J774 cells(American Type Culture Collection) were seeded at a density of 1×10⁶host cells in 25 cm² Corning tissue culture flasks. The cells wereinfected with 5×10⁷ Salmonella typhimurium #14028/ml DMEM culture mediumfor 1 hour, washed with fresh medium, and further cultured for 6 hourswith 50 μg/ml gentamicin added to the culture medium in order to killthe external but not the internalized Salmonella. The melanoma cellswere then harvested by scraping them from the substratum in isotonic 1mM EDTA solution. The cells were pelleted, resuspended in PBS and analiquot was removed for quantitation of the bacteria found within themelanoma cells. The remainder of the melanoma cells were assayed forβ-galactosidase activity.

Three Salmonella typhimurium clones were used: i) strain 14028 in whichβ-galactosidase was constitutively expressed; ii) strain 14028 in whichβ-galactosidase was expressed through activation of the pagB promoter;iii) strain 14028 in which β-galactosidase was expressed throughactivation of the pagC promoter. Thus, through measurements ofβ-galactosidase activity, analyses of bacterial pagB and pagC promoterinduction in melanoma cells were carried out. The results are detailedin Table 14.

TABLE 14 ENHANCED EXPRESSION OF BACTERIAL PROMOTERS PAGB AND PAGC INCULTURED HUMAN MELANOMA M2 CELLS⁺ Promoter-Induced Activity: ConstituteActivity Cell Line pagB pagC human epithelial 1.3:1 7.2:1  mousetransformed macrophage 2.9:1 17:1 human melanoma 3.8:1 31:1 *relativeactivation of pagB and pagC was assessed through expression ofpromoter-inducible β-galactosidase activity.

Both the pagB and pagC Salmonella promoters were induced in humanmelanoma cells. Levels of induction in melanoma cells were greater thanseen in either the epithelial or macrophage cell lines. These datademonstrate that the pagB or pagC promoter could be used to expressgenes, such as HSV TK or E. coli cytosine deaminase, in a melanomacell-specific manner.

13. EXAMPLE Cloning and Expression of Prodrug Converting Enzymes

The following sections demonstrate useful systems for expression ofprodrug-converting enzymes useful for the methods and compositions ofthe present invention.

13.1. Cloning and Expression of Herpes Simplex Virus Thymidine Kinase inSalmonella typhimurium

Herpes simplex thymidine kinase (HSV TK) is known to be an effectivepro-drug converting enzyme in the inhibition of melanoma tumor growth(Bonnekoh et al., 1995, J. Invest. Derm. 104:313-317). Accordingly,procedures were carried out to insert an HSV TK gene with theβ-lactamase signal sequence into both Salmonella typhimurium wild typestrain 14028 and super-infective tumor-specific mutant clone 72 which isderived from the wild type strain.

Herpes Simplex Thymidine Kinase Cloning By PCR

Plasmid DNA of the vector pHETK2 (Garapin et al., 1981, Proc. Natl.Acad. Sci. USA 78:815-819) was prepared by alkaline lysis,phenol/chloroform extraction and ethanol precipitation. PCR primersbased on the complete sequence for the Herpes simplex thymidine kinase(McKnight, 1980, Nuc. A. Res. 8:5949-5964) were: forward5′-GATCATGCATGGCTTCGTACCCCGGCC-3′ (SEQ ID NO:1) and reverse5′-CTAGATGCATCAGTGGCTATGGCAGGGC-3′, (SEQ ID NO:2) which corresponds tobases 310-328 (forward) and 1684-1701 (reverse) of the publishedsequence, with an added sequence of GATCATGCAT (portion of SEQ ID NO:1)or CTAGATGCAT (portion of SEQ ID NO:2) (NsiI site and spacer) at the 5′end of each primer. Each reaction mixture contained 50 ng DNA template,10 pmoles of each primer, 100 mM deoxynucleotide triphosphates, 1.5 mMMg⁺⁺ and 0.5 unit's. Taq polymerase (Perkin Elmer Cetus, Norwalk,Conn.). Amplification was performed by 35 cycles of 94° C. for 1 minute;50° C. for 15 seconds; 55° C. for 1 minute; and 72° C. for 2 minutes.The amplified DNA was purified and was cloned into either pBluescript IIKS+ and sequenced with T3 and T7 primers to confirm the correct DNA hadbeen cloned or was cloned into p279 cut with Pst1 which provides theβ-lactamase signal sequence (Talmadge et al., 1980, Proc. Natl. Acad.Sci. USA 77:3369-3373). Transformants were screened using a probegenerated from the original template by random priming (BoehringerMannheim, Indianapolis, Ind.) using [α-³²P]dCTP. Positive clones werefurther screened by immunoblot.

SDS-PAGE and Immunoblot

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) wasperformed on bacterial lysates according to Weber and Osbom, 1975,Proteins and sodium dodecyl sulfate: Molecular mass determination onpolyacrylamide gels and related procedures. In: H. Neurath and R. Hill(eds) The Proteins, Third Edition, vol. 1, Academic Press, New York pp.179-223. Immunoblots were performed according to Towbin et al., 1979,Proc. Natl. Acad. Sci. USA 76:4350-4354. Primary anti-TK antibodies weregenerally used at a 1:1000 dilution. Secondary anti-mouse antibodieswere alkaline phosphatase-conjugates (Promega, Madison, Wis.) used at a1:7,500 dilution, followed by nitroblue tetrazolium (NBT) and5-bromo-4-chloro-indolyl phosphate (BCIP) colorimetric detection(Promega).

Thymidine Kinase Assay

Bacterial lysates were prepared by pelleting 1 ml of log-phase bacterialculture for 30 seconds at 12,000×g in a microfuge centrifuge. The pelletand supernatant were retained separately and the supernatant was furthercleared by centrifugation for 10 min at 12,000×g. The pellet was furthertreated by resuspension in 100 μl of phosphate buffered salinecontaining 1 mg/ml lysozyme and 1% (v/v) Triton X-100 and subjected tothree cycles of rapid freezing and thawing. The resulting material wasclarified by centrifugation at 12,000×g for 2 minutes. Thymidine kinaseactivity was assayed using a modified version of the assay described bySummers and Summers, 1977, J. Virol. 24:314-318. The reaction mix wasincubated at 37° C. for 1 hour and then bound to DE81 paper (Whatman),washed, and the associated radioactivity determined in a gamma counter.

Salmonella Transformation

Transformation of Salmonella strains was performed by electroporation asdescribed by O'Callaghan and Charbit, 1990, Mol. Gen. Genet.223:156-158. Plasmids transfected into Salmonella included pHETK2(Garapin et al., 1981, Proc. Natl. Acad. Sci. USA 78:815-819) p279(Talmadge et al., 1980, Proc. Natl. Acad. Sci. USA 77:3369-3373) and twoindependent isolates of β-lactamase fusions, p5-3 and p21A-2 (See FIG.4-C for a diagram of p5-3 and p21A-2 where these plasmids are designated“pTK Sec 1”. Salmonella typhimurium strains transfected were the wildtype 14028 and the super-infective clone 72.

Two independent β-lactamase-TK gene fusion constructs were isolated andexpressed in Salmonella typhimurium 14028 wild type and clone 72. Animmunoblot analysis and corresponding enzyme activity assay arepresented in FIGS. 4A and 4B. All three TK-containing vectors, thecytoplasmically expressed pHETK2 and the β-lactamase fusions p5-3 andp21A-2, were detectable by immunoblot and enzyme assay. Relativelylittle enzyme activity was recovered from the culture supernatants.Since the immunoblot analysis shows processing of the signal sequence,secretion into the periplasmic space of the Salmonella typhimurium isexpected.

13.2. Systems For Expression of Herpes Simplex Thymidine Kinase UsingVarious Promoters and Secretion Signals

A number of constructs were made to express TK using other promoters andother secretion signals.

13.2.1. Expression as a Staphylococcus Protein a Fusion Under the LaciPromoter

Herpes simplex thymidine kinase was amplified by PCR as described inSection 13.1 above and cloned into the Pstl site of pBluescript. This TKclone was subcloned from bluescript to the BamHI and HindIII cite of thesecretion vector pEZZ18 (Promega, Madison, Wis.; Nilsson and Abrahamsen,1990, Methods in Enzymology 185:144-161). This resulted in an in-framefusion with Staphylococcus protein A under the lacI promoter. Thisplasmid was designated pTK-Sec2 and is diagramed in FIG. 4-C. PlasmidpTK-Sec2 expresses thymindine kinase as determined by an immunoblot.

13.2.2. Cloning of the Serratia Marcesens Chitinase Signal Sequence andPromoter

The promoter and signal sequence of Serratia marcesens chitinase I(Jones et al., 1986, EMBO J. 5:467-473) was cloned by PCR. The forwardand reverse primers had the following sequence:CTAGACTAGTTTGTCAATAATGACAACACCC (forward) (SEQ ID NO:3) andGATCGGATCCTTGCCCGGCGCGGCGGCCTG (reverse) (SEQ ID NO:4) which containSpeI and BamHI sites, respectively. The resulting product was clonedinto pSP72 and confirmed by DNA sequencing. This plasmid was designatedpSP-CHT and is also diagramed in FIG. 4-C.

13.2.3. Expression as a Chinase Signal Sequence Fusion Under the Controlof the Chitinase Promoter

Herpes simplex thymidine kinase in pBluescript was subcloned into thepSP-CHT vector using BamHI and HindIII. This results in an in-framefusion with the chitinase signal sequence under the chitinase promoter.This plasmid was designated pTK-Sec3 and is also diagramed in FIG. 4-C.Plasmid pTK-Sec3 expresses thymindine kinase as determined by animmunoblot.

13.3. Expression of P450 Oxidoreductase prodrug Converting Enzyme inBacteria Using an Exogenously Inducible Promoter

The sulA promoter element (GENBANK #V00358; Cole 1983) was cloned fromE. coli genomic DNA by PCR using forward(CTAGAAGCTTATAAGGGTTGATCTTTGTTGTC) (SEQ ID NO:5) and reverse(GTACGATATCCAGAACGATGTGCATAGCCTG) (SEQ ID NO:6) primers whichincorporate the HindIII and EcoRV restriction sites respectively. ThePCR conditions were 35 cycles of 95° C., 1 minute; 55° C. 1 minute; and72° C. for 1 minute. The product was cloned into pSP72 and sequencedwith the T7 primer to confirm that the correct DNA had been obtained.The cloned DNA fragment was 100% identical to the published sequence.

The NADPH-dependent cytochrome p450 oxidoreductase (p450 OR) CDNA clonein the EcoRI site of pBluescript that lacks the first initiating ATG(deletion of the first 11 nucleotides) of the cDNA described by Yamanoet al., 1989, Molecular Pharmacol. 35:83-88, was fused with the sulApromoter and initiating sequence by cloning the sulA promoter obtainedas described above into the HindIII and EcoRV site of the p450oxidoreductase gene. The resulting fusion consists of the sulA promoter,including the sulA ATG (methionine) and subsequent 9 amino acids(YTSGYAHRS) (SEQ ID NO:7) as well as six amino acids which followintroduced from the DNA polylinker and PCR primers (SGYRIP) (SEQ IDNO:8) followed with the second amino acid of p450 OR, which is G. Thisconstruct, pSP-SAD4-5 is diagramed in FIG. 4-D.

13.4. Effect of Expression of P450 Oxidoreductase Conversion of ProdrugOn Bacterial Growth

It has been previously shown (Shiba et al., 1959, Nature 183:1056-1057)that some strains of bacteria are sensitive to low levels of mitomycin.Therefore, to compare the sensitivity of a specific bacterial strainwith and without the sulA::p450 OR expression plasmid the followingexperiment was performed. If the construct is functional, the presenceof the p450 OR gene is expected to result in increased activation ofmitomycin resulting in decreased growth of the bacteria. The sulA::p450OR expression plasmid used in this experiment is similar to pSP-SAD4-5(Section 13.3) except that it is in a pbluescript (pBS) backbone and hasthe β-galactosidase transcription unit. This construct is also diagramedin FIG. 4-D. The pBS plasmid, with and without the expression construct,was transfected into Escherichia coli DH5α by electroporation and clonescontaining the correct plasmids were obtained and confirmed by plasmidisolation and DNA restriction analysis. For each of the twoplasmid-bearing strains, a fresh, 4 hr (late log) culture was diluted1:100 into LB with 100 μg/ml ampicillin to select for the presence ofthe plasmid and grown at 37° C. at 250 rpms. Mitomycin C was added tothe cultures in amounts of 0.0, 0.1 and 0.5 μg per ml.

Optical density was determined at 600 nm using a Perkin Elmer doublebeam spectrophotometer at 2 and 18 hour time points. The results arepresented in Table 14(A).

TABLE 14A GROWTH OF BACTERIAL CULTURES IN THE PRESENCE OF MITOMYCIN COD₆₀₀ t = 2 hours post drug Amount of Mitomycin C (μg/ml) added Plasmid0 0.1 0.5 pBS 0.052 0.050 0.053 sulA::p450 OR 0.037 0.030 0.024 OD₆₀₀ t= 18 hours post drug Mitomycin C (μg/ml) added Plasmid 0 0.1 0.5 pBS2.33 2.23 1.93 sulA::p450 OR 2.26 0.34 0.071

Comparison of the growth of the E. coli strain DH5α containing pBS andsulA::p450 OR in the absence of drug at the 2 and 18 hour time pointsshows that the presence of the construct partially inhibits the rate ofgrowth but does not inhibit attaining a high final OD at 18 hours. Thesedata also show that bacteria carrying the pBS backbone plasmid alone areonly partially inhibited at the higher mitomycin concentration. However,those carrying the sulA::p450 construct show significant inhibition atboth early and late time points at both mitomycin concentrations. Thesedata indicate a strong dose response to mitomycin conferred by thepresence of the sulA::p450 construct.

13.5. Expression of Cytosine Deaminase in Salmonella typhimurium

E. coli cytosine deaminase (CD) has been shown to be an effectiveprodrug-converting enzyme useful for gene therapy (Hirschowitz et al.,1995, Human Gene Therapy 6:1055-1063; Huber et al., 1993, Cancer Res.53:4619-4626; Huber et al., 1994, Proc. Natl. Acad. Sci. USA91:8302-8306; Moolten, 1994, Cancer Gene Ther. 1:279-287; Mullen et al.,1992, Proc. Natl. Acad. Sci. USA 89:33-37; Mullen et al., 1994, CancerRes. 54:1503-1506; Trihn et al., 1995, Cancer Res. 55:4808-4812). CDfunctions by converting the non-toxic 5-fluorocytosine (5-FC) to thetoxic compound 5-fluorouracil (5-FU). Salmonella possess an endogenousCD, however, its expression is catabolite repressed (West and O'Donovan,1982, J. Bacteriol. 149:1171-1174). A CD expression vector using theconstitutively active β-lactamase promoter to ensure expression of CDwithin tumors was cloned as described below.

Cloning and Expression of CD

PCR primers based on the complete sequence for E. coli cytosinedeaminase (Huber et al., 1993, Cancer Res. 53:4619-4629) were forward:5′-GATCATGCATGTGGAGGCTAACAGT-3′ (SEQ ID NO:9) and reverse:5′-CTAGATGCATCAGACAGCCGCTGCGAAGGC-3′ (SEQ ID NO:10), corresponding tothe published sequence, with the added sequence GATCATGCAT (portion ofSEQ ID NO:9) or CTAGATGCAT (portion of SEQ ID NO:10) which is a NsiIsite and spacer at the 5′ end of each primer. Each 25 μl reactionmixture contained 50 ng DNA template, 10 pmoles of each primer, 100 mMdeoxynucleotide triphosphates, 1.5 mM Mg and 0.5 units Taq polymerase(Perkin Elmer Cetus, Norwalk, Conn.). Amplification was performed by 35cycles of 94° C. for 1 minute; 50° C. for 15 seconds; 55° C. for 1minute; and 72° C. for 2 minutes. The amplified DNA was purified on anagarose gel and the band of correct size was cloned into 1) pBluescriptII KS+ and sequenced with T3 and T7 primers to confirm the correct DNAhad been cloned and 2) p279 cut with Pstl which provides the β-lactamasesignal sequence and the constitutive β-lactamase promoter. This secondconstruct was designated pCD-Sec1 and is diagramed in FIG. 4-E.Transformants were screened using a [α-³²P] dCTP-labeled oligonucleotideprobe. Positive clones were further screened by immunoblot using anti-CDantibodies described below.

Primary Antibodies to Cytosine Deaminase

CD was subcloned from pBluescript into PGEX I and expressed using IPTG.The expressed protein was found to be insoluble and present in inclusionbodies. CD-glutathione-S-transferase (GST) fusion protein was purifiedfrom inclusion bodies by washing in 0.1% w/v Triton X-100, repelletedand resuspended in SDS-PAGE sample buffer. The material was separated ona 3 mm preparative 10% polyacrylamide gel and excised aftervisualization with 3M potassium acetate at 4° C. The purifed bands werehomogenized and injected i.p. into DBA2J mice with Freund's complete(day 0) and incomplete (day 14) adjuvant. After 6 weeks the mice werebled and the ability of the serum antibodies to bind to cloned CDconfirmed by Immunoblot.

SDS-PAGE and Immunoblot

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed onbacterial lysates according to Weber and Osbom, 1975, Proteins andsodium dodecyl sulfate: Molecular mass determination on polyacrylamidegels and related procedures. In:H. Neurath and R. Hill (eds) TheProteins, Third Ed., Vol. I, Academic Press, New York, pp. 179-223.Immunoblots were performed according to Towbin et al., 1979, Proc. Natl.Acad. Sci. USA 76:4350-4354. Primary anti-CD antibodies described abovewere generally used at a 1:500 dilution. Secondary anti-mouse antibodieswere alkaline phosphatase-conjugates (Promega, Madison, Wis.) used at a1:7,500 dilution, followed by nitroblue tetrazolium (NBT) and5-bromo-4-chloro-indolyl phosphate (BCIP) calorimetric detection(Promega, Madison, Wis.).

CD Enzyme Assay

Bacterial lysates were prepared by pelleting 50 ml of overnightbacterial culture at 3000×g for 10 minutes and resuspending them in 2.5ml of PBS. The cells were sonicated and the debris removed by pelletingin a microfuge at 12,00×g for 10 minutes. The enzyme assay performed wasmodified from Mullen et al., 1992, Proc. Natl. Acad. Sci. USA:89:33-37.10 μl of cell extract was incubated with 1 μl [H³]-5FC (1 μCI/μl) a 37°C. 1 μl was spotted on a Kodak 13254 microcrystalline nitrocellulose TLCplate, Eastman Kodak, Rochester N.Y., and separated using 95:5Butanol:water with unlabeled 5FC and 5FU markers. The plates were cutbased upon separation of the marker lanes and quantified using a liquidscintillation counter.

Salmonella Transformation

Transformation of Salmonella strains was performed by electroporation asdescribed by O'Callaghan and Charbit, 1990, Mol. Gen. Genet.223:156-158. Plasmids transfected into Salmonella were p279 andpCD-Sec1. Salmonella typhimurium strains transfected were strains YS721,YS7211, YS7212 and YS7213 which are described infra in Section 18. E.coli strains transfected were strains DH5α and KL498 (Δcod).

Biodistribution of Salmonella Carrying the CD Expression Construct

Salmonella typhimurium clone YS7212 carrying the CD expression constructpCD-Sec1 was grown in LB media to an OD₆₀₀ of 0.8. An aliquot of 1.0×10⁶bacteria were inoculated i.p. into C57/B6 mice which had been implantedwith 2×10⁵ B16 melanoma cells 16 days prior to the bacterial infection.At two days post bacterial infection, mice were sacrificed and tumorsand livers assayed for the presence of the bacteria by homogenizationand plating of serial dilutions.

The expressed protein product of pCD-Sec1 bound to the anti-CDanti-serum by immunoblot analysis. When this clone was transferred tothe E. coli strain KL498 which lacks CD, it was found to confer a highdegree of enzyme expression as determined by the conversion of 5FC to5FU, as shown in FIG. 4-F. FIG. 4-F also demonstrates that the clonedCD-expression plasmid gives higher levels of conversion than the E. colistrain MG1655 which expresses the wild type haploid cod gene whichencodes for endogenous CD.

14. EXAMPLE Proliferation of Salmonella typhimurium Clone #72⁵⁻³⁻² inMelanoma Tumors in Mice

In a similar set of experiments as in Section 10.2, DBA/2J mice(approximately 10 weeks) were inoculated (s.c.) with 3×10⁵ Cloudman S91melanoma/macrophage hybrid #48 cells in each of 4 sites over the rightand left shoulders and flanks. Tumors were palpable 10-12 days postinoculation of tumor cells from tissue culture into mice. After twoweeks post-inoculation of tumor cells (s.c.), tumor-bearing mice wereadditionally inoculated (i.p.) with 2×10⁵ c.f.u. of S. typhimurium clone72 containing the HSV TK gene with the β-lactamase signal sequence whichis designated 72⁵⁻³⁻². After 2 and 10 days of bacterial infectionwithout antibiotic treatment, representative tumor-bearing animals weresacrificed and their tumors and livers were homogenized and quantitatedfor c.f.u. of Salmonella per gram of tissue. In addition, individualclones of bacteria were isolated from the liver and tumor homogenates 10days post-infection and tested for the genetic markers xyl^(neg)(inability to metabolize xylose, characteristic of clone #72) andtet^(res) (resistance to the antibiotic tetracycline). The genotype ofthe inoculated Salmonella typhimurium clone #72⁵⁻³⁻² was xyl^(neg) andtet^(res). The tet^(res) clones of Salmonella were assumed to carry theHSV TK gene, since the HSV TK gene was carried on a plasmid that carriedthe tet^(res) marker.

The results are presented in Tables 15 and 16. After 2 days ofinfection, the tumors contained an average of 1.5×10⁹ Salmonella/g tumorand 2.0×10⁵ Salmonella/g liver, with an average ratio of tumor:liver ofabout 7,500:1.

TABLE 15 DISTRIBUTION OF SALMONELLA TYPHIMURIUM 2 DAYS FOLLOWINGINOCULATION (I.P.) INTO CLOUDMAN S91 MELANOMA-BEARING DBA/2J MICE TissueSalmonella/g Tissue Tumor/Liver Liver (n = 2) 2.0 × 10⁹ — Tumor (n = 4)1.5 ± 0.9 × 10⁹ 7,500:1

After 10 days of infection (Table 16), the tumors contained an averageof 2.9×10⁹ Salmonella/g tumor and 2.7×10⁵ Salmonella/g liver, a ratio of11,000:1 (tumor:liver), similar to the distribution of bacteria seen 1-2days post-infection. These results demonstrate that once inoculated(i.p.) into tumor-bearing mice, Salmonella enter the circulatory system,infect the tumor cells, proliferate within the tumors, and exist therein a compartmentalized fashion.

TABLE 16 DISTRIBUTION OF SALMONELLA TYPHIMURIUM 10 DAYS FOLLOWINGINOCULATION (I.P.) INTO CLOUDMAN 691 MELANOMA-BEARING DBA/2J MICE TissueSalmonella/gm Tissue Tumor/Liver xylose tetracycline Liver 2.7 × 10⁵ —neg  9/10 res Tumor #1 4.2 × 10⁹ 16,000:1 neg 3/7 res #2 3.1 × 10⁹12,000:1 neg 7/8 res #3 1.3 × 10⁹  4,800:1 neg 8/8 res Average 2.9 × 10⁹11,000:1

The results further demonstrate that 10 days post-infection, all of thebacterial clones examined were xyl^(neg), proving their geneticrelationship to the inoculated clone #72⁵⁻³⁻², and that 27/33 clonesremained tet^(res), demonstrating high degree of retention (82%) of theHSV TK containing plasmid within the host bacteria. In experiments notshown here, the same plasmid was found to be 100% retained after 42hours of infection in tumor-bearing mice.

In a continuation of the above experiments summarized in Tables 14, 15and 16, the Salmonella infections in melanoma-bearing mice werecontinued for a total of 4 weeks. To alleviate the symptoms ofSalmonella poisoning (shaking, matted hair) the animals were placed onantibiotics for the final two weeks. Such antibiotic treatmentsconsisted of the inclusion in the mouse drinking water of SULFATRIM™Pediatric Suspension (Schein Pharmaceutical, Inc.; sulfamethoxazole 40mg/ml, trimethoprim 8 mg/ml) at a final concentration of 15 mlSULFATRIM™/500 ml drinking water. At termination of the experiment, thesurviving mice were sacrificed by euthanasia, and the tumors and liverswere removed. Portions of the tissues (1-2 mm³) were fixed in formalinand stained for histological examination. The remaining portions wereweighed, homogenized in 5 ml LB broth/g tissue, and the number ofSalmonella were quantitated on LB agar plates. Results are presented inTable 17.

TABLE 17 DISTRIBUTION OF SALMONELLA TYPHIMURIUM 4 WEEKS FOLLOWINGINOCULATION (I.P.) INTO MELANOMA-BEARING DBA/2J MICE Tissue Salmonella/gtissue Tumor/Liver Liver 5.1 × 10⁷ — Tumor #1 2.2 × 10⁹ 43:1 #2 9.4 ×10⁹ 18:1 #3 2.3 × 10⁹ 45:1 #4 2.0 × 10⁸  6:1 Average 1.4 ± 1 × 10⁹ 28:1

The excised melanoma tumors averaged less than 1 gram in weight comparedto 5-10 gm tumors in control animals at death (data not shown). It wasfound that these tumors contained an average of 1.4×10⁹ Salmonella/gtumor, similar to the number of tumor-infecting bacteria seen at 1, 2,and 10 days post-inoculation. However, the number of Salmonella in theliver increased during the 4 week infection, so that the average ratioof bacteria in tumor over liver was reduced to 28:1 compared to theratios obtained with infection periods of up to 10 days as seen inTables 14, 15 and 16.

15. EXAMPLE Microscopic Detection of Salmonella typhimurium in Melanomasin Vivo

15.1. Detection of Salmonella typhimurium Within Cloudman S91 MelanomasGrowing in DBA/2J Mice

In order to study the histopathology of Salmonella infection in thetumor-bearing mice, representative melanoma tumors were removed fromeuthanized mice with or without Salmonella infection. Portions of thetissues (1-2 mm³) were fixed in formalin, embedded and sectioned, andthe sections stained with either hematoxylin and eosin, or tissue gramstain for histological examination. Results of these studies are shownin FIGS. 5A-B.

FIGS. 5A-B are photomicrographs of histological sections from a CloudmanS91 melanoma/macrophage hybrid #48 melanoma growing subcutaneously in aDBA/2J mouse. The tumor was excised from a mouse that had beeninoculated 2 days earlier with 3×10⁵ c.f.u. Salmonella typhimuriumsuper-infective clone 72 carrying the HSV TK gene, 72⁵⁻³⁻². A portion ofthe tumor was weighed, immersed in LB at 5 ml/g tumor, homogenized witha ground glass homogenizer, and the tumor homogenate was plated ontoLB-Agar culture plates at various dilutions in order to quantitate theamount of Salmonella typhimurium in the tumor. Quantitation of thebacteria revealed that the tumor contained 1.4×10⁹ Salmonella/g. FIG.5A. A section stained with hematoxyn and eosin shows a cross-section ofthe tumor with an area of necrosis, denoted by the arrow. FIG. 5B. Asection stained with tissue gram stain shows gram negative bacteria inan area of necrosis area of the tumor. When viewed with the lightmicroscope, the bacteria stain pink/purple against a yellow background.Salmonella-infected necrotic areas were surrounded with dead tumor cellsthat did not stain with tissue gram stain but which could be detectedthrough melanin-containing melanosomes (see FIG. 6). These results showthat the necrotic areas of solid tumors are accessible to Salmonellawhen the bacteria are introduced into a tumor-bearing host via thecirculatory system.

In an additional set of analyses, sections of Cloudman S91melanoma/macrophage hybrid #48 melanoma tumors growing in aSalmonella-infected mouse were examined with the electron microscope. Toinitiate the experiment, a mouse was inoculated s. c. with 8×10⁵ tumorcells. A palpable tumor mass was detected 11 days later, at which timethe mouse was inoculated i.p. with 3.6×10⁶ c.f.u. of S. typhimuriumsuper-infective clone #72. Forty-two hours post-inoculation, the mousewas sacrificed by metofane anesthesia. The tumor was excised usingaseptic techniques. Quantitation of the bacteria within the tumorrevealed that the tumor contained approximately 7.5×10⁹ S. typhimurium/gupon excision at 42 hours. In contrast, the concentration of S.typhimurium in the liver from the same mouse was approximately2.0×10⁷/g, a ratio of bacteria in tumor to liver of approximately 400:1.

A second portion of the tumor was cut into 1-2 mm³ pieces and fixed in ½strength Karnovsky's fixative for 6 hours at 4° C., followed by washingin cacodylate buffer overnight. The tumor tissue was post-fixed with 1%OsO₄ and 1.5% potassium ferrocyanide in cacodylate buffer for 2 hoursand embedded in Spurr's resin. Ultrathin sections were stained withuranyl acetate and lead citrate. They were viewed with a Zeiss 109electron microscope.

Shown in FIG. 6 is an electron micrograph of a field within a melanomatumor that includes two separate S. typhimurium along with numerousmelanosomes, which are specialized subcellular organelles present in thecytoplasm of melanoma cells. The presence of bacteria along with themelanosomes provides proof that the S. typhimurium entered the cytoplasmof the melanoma cell via the bloodstream of the mouse. The S.typhimurium in the electron micrograph appear identical to those shownpreviously. in intestinal epithelial cells following an experimentalinfection of the mouse, Takeuchi, 1967, Am. J. Pathol. 50:109-1361.

In summary, i) examination with the light microscope revealed thatSalmonella typhimurium exists in the necrotic areas of Cloudman S91melanomas growing in infected DBA/2J mice; and ii) examination with theelectron microscope revealed that Salmonella typhimurium also existswithin the cytoplasm of melanoma tumor cells.

15.2. Distribution of Salmonella typhimurium Within Mouse B16F10Melanoma Tumors Grown In C57BL/6J Mice

C57BL/6J mice (11-13 weeks old) were inoculated s.c. in two sites(shoulder and flank), with 3.5×10⁵ B16F10 mouse melanoma cells per site.After the appearance of palpable tumors (approximately 2 weeks) theanimals were further inoculated i.p. with about 10⁵ bacteria of thefollowing three strains: i) wild type Escherichia coli K-12 strain #CSH101; ii) Salmonella typhimurium strain 14028; and iii) mutant Salmonellatyphimurium super-infective clone 72 carrying the HSV thymidine kinasegene, 72⁵⁻³⁻². After about 2 days of infection, mice were euthanized byanesthesia with metofane. Tumors and livers were removed aseptically,rinsed with sterile 0.9% NaCl, weighed, and homogenized in LB broth at aration of 5:1 (vol. broth:wt. tumor). Prior to homogenization, 1-2 mm³pieces of tissue were removed from representative tumors, fixed with ½strength, Karnovsky's fixative, and processed for analysis with theelectron microscope. Bacteria in the homogenates were quantitated byplating onto LB plates, incubating overnight at 37° C., and countingbacterial colonies.

Results were as follows: i) wild type E. coli were found in relativelylow numbers in both the tumor and liver of the inoculated animals atconcentrations averaging <10³/g tumor and <10²/g liver. ii) wild type S.typhimurium were found in significantly higher numbers than E. coli inboth tumor and liver, with infecting bacteria ranging from 2×10⁷ to6×10⁸ c.f.u./g tumor, and 4×10⁶ c.f.u./g liver. One of the two C57BL/6Jmice inoculated with the wild type S. typhimurium strain died, possiblyfrom septic shock. iii) S. typhimurium super-infective clone 72 werealso found in significantly higher numbers than E. coli in both tumorand liver, further, the number of clone 72 S. typhimurium/g liver wassignificantly lower than the number of wild type S. typhimurium/g liver.The results are detailed below in Table 18.

TABLE 18 DISTRIBUTION OF SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI 2DAYS FOLLOWlNG INOCULATION (I.P.) INTO C57B6 MICE BEARING B16F10 TUMORSBacteria/ Bacterial Strain Mouse Tissue gm Tissue Tumor/Liver E. coliK-12 A Liver 355 — (CSH #101) Tumor #1 1200 4:1 Tumor#2 50 1:7 A′ Liver100 — Tumor#1 50 1:7 S. typhimurium (14028 B Liver 4.3 × 10⁶ — wildtype) Tumor #1 2.3 × 10⁷ 5:1 Tumor #2 6.0 × 10⁸ 136:1 B′ (dead) — — S.typhimurium (clone C Liver 2.0 × 10⁴ — #72⁵⁻³⁻²) Tumor #1 1.0 × 10⁸5,000:1 Tumor #2 1.2 × 10⁵ 6:1 C′ Liver 8.5 × 10⁴ — Tumor #1 9.3 × 10⁸11,000:1 (2.5 g)

In summary, Salmonella typhimurium displays natural capabilities overEscherichia coli in its ability to infect and proliferate within B16F10melanoma tumors. Furthermore, super-infective clone 72⁵⁻³⁻² displayssuperior qualities to its wild type parental strain 14028 in its reducedinfection of liver in C57BL/6J mice, i.e., the wild-type strain 14028showed greater infectivity toward liver than did clone 72⁵⁻³⁻² Thehigher infectivity of liver by the wild-type Salmonella was consistentwith the observed greater lethality of the wild type stain toward DBA/2Jmice and the greater infectivity of liver in DBA/2J mice than thatproduced by clone 72 as seen in Table 11B. Together, the results inTables 11B and 18 provide the first evidence that selection for strainsof bacteria or other parasites with enhanced tumor specificity in vitrocan yield mutant strains with attenuated host toxicity in vivo.

15.3. Microscopic Detection of Salmonella typhimurium Within B16F10Melanomas Growing in C57BL/6J Mice

Representative B16F10 melanoma tumors were removed from euthanized micewith or without Salmonella infection. Portions of the tissues (1-2 mm³)were fixed informalin, embedded and sectioned, and the sections stainedwith either hematoxylin and eosin, or tissue gram stain for histologicalexamination. Results of these studies are shown in FIGS. 7A-B. FIGS. 7Aand 7B are light micrographs of histological sections from a B16F10melanoma growing subcutaneously in a C57BL/6J mouse. The tumor wasexcised from a mouse that had been inoculated 2 days earlier with 2×10⁵c.f.u. Salmonella typhimurium super-infective clone, 72⁵⁻³⁻² carryingthe HSV TK gene. Quantitation of the bacteria within the tumor revealedthat the tumor contained approximately 9×10⁸ c.f.u. S. typhimurium/gupon excision 2 days post-infection with bacteria. In contrast, theconcentration of S. typhimurium in the liver from the same mouse wasapproximately 2.0×10⁵/g, a ratio of bacteria in tumor to liver ofapproximately 400:1. FIG. 7: A section stained with tissue gram stainshows gram negative bacteria in an area of necrosis within the tumor.The infected necrotic area is surrounded by dead melanoma cells that donot stain with the tissue gram stain but which appear brown in color dueto the presence of melanized melanosomes. When viewed with the lightmicroscope, the bacteria stain pink/purple against a yellow background.The results show that necrotic areas of B16 melanoma tumors areaccessible to Salmonella when the bacteria are introduced into atumor-bearing host via the circulatory system.

A second portion of the above-described tumor was cut into 1 mm³ piecesand fixed in ½ strength Karnovsky's fixative for 6 hours at 4° C.,followed by washing in cacodylate buffer overnight. The tumor tissue waspost-fixed with 1% OsO₄ and 1.5% potassium ferrocyanide in cacodylatebuffer for 2 hours, and embedded in Spurr's resin. Ultrathin sectionswere stained with uranyl acetate and lead citrate. They were viewed witha Zeiss 109 electron microscope as depicted in FIG. 8.

The electron micrograph in FIG. 8 shows numerous Salmonella typhimuriumin extracellular spaces, denoted by arrows, in an area of necrosis. Asingle bacterium is also seen within the cytoplasm of a dying melanomacell. The cytoplasm of the dying melanoma cell also contains numerousblack melanosomes, characteristic of the B16F10 melanoma.

The S. typhimurium in the electron micrograph appear identical to thoseshown previously in intestinal epithelial cells following anexperimental infection of the mouse, Takeuchi, 1967, Am. J. Pathol.50:109-136.

In summary, i) examination with the light microscope revealed thatSalmonella typhimurium exist abundantly in the necrotic areas of B16F10melanomas growing in infected B16F10 mice; and ii) examination with theelectron microscope revealed that Salmonella typhimurium also existwithin the cytoplasm of tumor cells. Salmonella were also observed intumor-associated neutrophils.

16. EXAMPLE Use of Super-infective Tumor-specific Gene-deliveringSalmonella typhimurium For Treatment of Mice Bearing Melanoma Tumors

16.1. Treatment of Cloudman 591 Melanoma

Salmonella typhimurium super-infective mutant 72⁵⁻³⁻², constitutivelyexpressing the Herpes simplex virus thymidine kinase gene with theβ-lactamase signal sequence, was used for gene therapy of melanoma inmice (see FIG. 4C). DBA/2J mice (approximately 10 weeks) were inoculated(s.c.) with 3×10⁵ Cloudman S91 melanoma/macrophage hybrid cells in eachof 4 sites over the right and left shoulders and flanks. Tumors werepalpable 10-12 days post-inoculation of tumor cells.

After two weeks post-inoculation of tumor cells, tumor-bearing mice werefurther inoculated (i.p.) with 2×10⁵ c.f.u. of S. typhimurium clone 72containing the HSV thymidine kinase gene with the β-lactamase secretorysignal sequence which is designated 72⁵⁻³⁻². Twelve hours afterinoculation of the bacteria, some of the mice were, further inoculated(i.p.) with 2.5 mg ganciclovir sodium (CYTOVENE™, ganciclovir sodiumSyntex Laboratories, Palo Alto, Calif.) in isotonic saline. These samemice received this dosage of ganciclovir four times over a 3 day period.Control tumor-bearing mice also received ganciclovir but no bacteria.Another set of tumor-bearing mice was inoculated with bacteria, butreceived no ganciclovir. At various times appropriate groups of mice,treated as above, were also given the antibiotic Sulfatrim™ PediatricSuspension (Schein Pharmaceutical, Inc.; sulfamethoxazole 40 mg/ml,trimethoprim 8 mg/ml) at a concentration of 15 ml Sulfatrim™/500 mldrinking water.

Results were as follows:

1) Control melanoma tumor-bearing mice, receiving ganciclovir andantibiotic treatment (Sulfatrim™ in drinking water) but no bacteria,developed rapidly growing tumors that initially doubled in size every 3to 4 days, determined by caliper measurements as shown in FIG. 9. Theseanimals exhibited little or no side-effects from the ganciclovirtreatment, confirming previous reports on the minimal toxicity of theganciclovir pro-drug in mice in the absence of a suitable thymidinekinase converting enzyme (Bonnekoh et al, 1995, J. Invest. Dermatol.104:313-317). By 30 days post-inoculation with tumor cells, all mice inthis group had formed massive subcutaneous tumors (5-10 gm) and had diedfrom melanoma.

2) One group of tumor-bearing mice received bacteria for a total of 10days without administration of antibiotics, and received no ganciclovir.These animals had tumors that were significantly reduced in size fromthe tumors in control mice (FIG. 10). The effect of Salmonella alone onreducing tumor size became evident several days after the effect ofSalmonella plus ganciclovir on tumors had been observed as describedbelow. However, all the animals in the “Salmonella alone” groupdeveloped symptoms of Salmonella infection (shaking, matted hair) and50% of these animals succumbed between 5-10 days post-infection. Theremaining animals were treated with the antibiotic Sulfatrim™ (ScheinPharmaceutical, Oral Suspension) at a concentration of 15 ml/500 mldrinking water. This treatment reduced the clinical symptoms ofSalmonella infection in the mouse population within 24-48 hours. Thesurviving animals from this protocol had significantly smaller tumorsthan control animals and remained alive past the 30 day period, when allof the control animals had died from melanoma.

3) Another group of tumor-bearing mice received ganciclovir plusbacteria during a 4-day treatment period. About 50% of the animalssuccumbed within 1-2 days of this treatment, apparently from theconversion of ganciclovir to its toxic, phosphorylated form by the HSVTK expressed by the Salmonella clone 72⁵⁻³⁻² within the body of themouse. At this time ganciclovir treatment was discontinued and thesurviving animals were placed on Sulfatrim™ antibiotic to control theSalmonella infection. The total time of exposure to Salmonella withoutantibiotic was 4 days. The survivors from this protocol hadsignificantly smaller tumors than control animals and remained alivepast the 30 day period when all the control animals had died frommelanoma (FIG. 11).

In a further set of experiments, tumor progression was measured withcalipers in various treated and untreated tumor-bearing mice. Groups ofmice bearing Cloudman S91 melanoma/macrophage hybrid #48 melanoma tumorsas described above were inoculated (i.p.) with 3×10⁵ c.f.u. Salmonellatyphimurium super-infective clone 72 carrying the Herpes simplex virusthymidine kinase gene, 72⁵⁻³⁻². Twenty-four hours after inoculation withbacteria, the mice were further inoculated with ganciclovir at doses of2.0 mg, with a total of 6 inoculations over a 5 day period. The micewere then subjected to antibiotic treatment with a combination of 15ml/500 ml SULFATRIM™ sulfa-based veterinary antibiotic and 20 μg/mlBAYTRIL™ enrofloxacin, a quinolone antibiotic (Miles) in their drinkingwater. BAYTRIL™ enrofloxacin, a quinolone antibiotic is1-cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylicacid. Tumor growth was assessed with periodic caliper measurements oftumor length, width, and height, and computed as tumor volume in mm³ bytechniques known to the science of tumor biology. Results were plottedon a semi-logarithmic scale and generation times, the time in hours forone doubling in volume, were calculated. The following formula was used:

Generation time=0.69(t)/In(T₁/T₂), where t equals the time in hoursbetween the initial tumor volume (T₁) and the final tumor volume (T₂)over the linear portion of the curve.

The results are shown in FIG. 9 and Table 19. Mean doubling times oftumors in untreated control mice and mice treated with ganciclovir butno Salmonella infection were similar, being 83 and 94 hoursrespectively. Tumors in mice treated with Salmonella for 5 days but noganciclovir doubled at a mean rate of 125 hours. Tumors in mice treatedwith Salmonella for 5 days as well as ganciclovir showed no growth overthe 10 day measurement period, and in some cases regressed with thetreatment.

TABLE 19 EFFECTS OF HSV TK-CONTAINING SALMONELLA TYPHIMURIUM ON THEGROWTH OF CLOUDMAN S91 MELANOMAS IN DBA/2J MICE ± TREATMENT WITHGANCICLOVIR Treatment Mean Tumor Doubling Time (hrs) none 83 ganciclovir94 S. typhimurium 125  S. typhimurium + ganciclovir no growth

In summary: a) Control tumor-bearing animals receiving ganciclovir andantibiotic treatment, but no Salmonella, succumbed from massive tumorswithin 30 days of inoculation of tumor cells; b) Animals receivingSalmonella alone followed by antibiotic treatment showed reduced tumorgrowth rate and prolonged survival over control animals; c) Animalsreceiving a combination of ganciclovir and Salmonella followed byantibiotic treatment showed little or no tumor growth compared tocontrol animals, and prolonged survival over control animals. Theresults indicate that Salmonella typhimurium expressing the Herpessimplex virus thymidine kinase gene was able to convert ganciclovir toits phosphorylated form within the melanoma tumors, thus reducing tumorsize and prolonging survival of the mice.

16.2. Treatment of B16F10 Melanoma

C57B6 mice were inoculated s.c., left shoulder region, with 5-105 B16F10melanoma cells from culture. At 8 days post-tumor implantation some ofthe mice were further inoculated i.p. with 2-10⁶ c.f.u. attenuatedSalmonella typhimurium strains YS721, YS7211, YS7212 or YS7213 (seeSection 18, infra) each carrying the HSV TK gene. At 11 days post-tumorimplantation, GCV (ganciclovir sodium, CYTOVENE™, Syntex Laboratories,Palo Alto, Calif.) was inoculated i.p. into groups of mice (n=5 or n=10)under the following treatment protocols: a) total dose=7.5 mg/mouse (2.5mg day 11, 1.25 mg day 12; 2.5 mg day 18, 1.25 mg day 19); b) totaldose=5.0 mg/mouse (2.5 mg day 11, 2.5 mg day 12); c) total dose=3.75mg/mouse (2.5 mg day 11, 1.25 mg day 12); d) total dose=2.5 mg/mouse(1.25 mg day 11, 1.25 mg day 12); e) total dose=1.25 mg/mouse (1.25 mgday 11). At 18 days post-tumor implantation (10 days post bacterialinoculation) all animals were given enrofloxacin antibiotic, 0.2 mg/ml,in their drinking water (BAYTRIL™) and maintained with this antibioticsupplement for 2 weeks. Tumor growth was assessed by calipermeasurements and computed as volume in mm³. Animals were euthanized andlisted as dead when the sum of their tumor measurements,length+width+height, reached 60 mm, or when they became moribund(listless, cessation of drinking).

The results obtained are illustrated in FIGS. 11C-H in Table 19(A).

TABLE 19(A) SURVIVAL OF C57B6 MICE INOCULATED WITH SALMONELLATYPHIMURIUM ± HSV TK GENE: EFFECTS OF GANCICLOVIR Time Treated/ControlStrain (n=) GCV Days ± S.D. T/C Control (10) -0- 25 ± 0 1.0 (10) 3.75 mg27 ± 1 1.1 YS7212 (10) -0- 42 ± 2 1.7 YS7212 (10) 3.75 mg 33 ± 4 1.3YS7212/p5-3 (10) -0- 45 ± 3 1.8 YS7212/p5-3 (10) 3.75 mg 40 ± 4 1.6YS7213 (10) -0- 34 ± 2 1.3 YS7213 (10) 3.75 mg 35 ± 2 1.4 YS7213/p5-3(10) -0- 29 ± 2 1.2 YS7213/p5-3 (10) 3.75 mg 33 ± 2 1.3 YS7211 (10) -0-40 ± 4 1.6 YS7211 (10) 3.75 mg 35 ± 4 1.4 YS7211/p5-3 (10) -0- 34 ± 21.4 YS7211/p5-3  (5) 1.25 mg 30 ± 4 1.2  (5) 2.50 mg 37 ± 4 1.5  (5)3.75 mg 38 ± 4 1.5  (5)  5.0 mg 42 ± 6 1.7  (5)  7.5 mg 39 ± 6 1.6 *Timeof death post tumor cell inoculation.

The results from the various treatment protocols for the B16F10melanoma-bearing mice were as follows:

1) Effects of GCV on Tumor-bearing Animals With no Bacterial Inoculation

Mice receiving melanoma cells but no bacteria were treated with GCV ondays 11-12 post inoculation with tumor cells at doses from 3.75 mg-10mg/mouse, depending on the experiment. In all trials, mice treated withGCV but no bacteria showed a small reduction in tumor volume that wasnoticeable within 5 days of GCV treatment and which persisted throughthe duration of the experiment, as shown in FIGS. 11C-E. GCV alsoelicited small but reproducible increases in survival time compared tothat of non-treated control animals, as outlined in Table 19(A). Theseeffects of GCV in the absence of bacterial treatment were not dependentupon dosage over the range studied.

These results demonstrate that the B16F10 cells employed in theexperiment might have had the capacity to convert GCV to its toxic,phosphorylated form. Consistent with such a notion, it was found thatproliferation of the B16F10 melanoma cells in culture was significantlysuppressed when GCV was supplemented to the culture medium at 25 μg/ml,but not at 10 μg/ml as shown in FIG. 11-F. Similar effects of GCV onDBA/2J mice bearing Cloudman S91 melanoma×macrophage hybrid 48, but notinoculated with bacteria, are reported in the Table 19.

2) Effects of GCV on Tumor-bearing Animals Treated With Bacteria NotContaining an HSV TK-plasmid

When tumor-bearing mice were inoculated with Salmonella strains YS7211,YS7212 and YS7213, none of which contained the HSV TK gene, and thentreated with GVC, GCV-mediated suppression of tumor growth was evident.Tumor suppression achieved with GCV was significantly greater than thatseen with the bacteria alone, even when the suppressive effect of GCV onB16F10 tumors in control animals was taken into account. This indicatedthat Salmonella typhimurium could convert GCV to its phosphorylated,toxic form without the HSV TK gene, perhaps through endogenousphosphotransferase enzymes (Littler, et al., 1992, Nature 358:160-162;Sullivan et al., 1992, Nature 358:362-364). Consistent with this notionwas the finding that in addition to suppressing tumor growth, somecombinations of bacteria and GCV treatment were highly toxic, shorteningsurvival times of the animals, shown in Table 19(A). Toxicity might haveresulted from production of phosphorylated GCV by those bacteria locatedin normal tissues such as liver or bone marrow.

3) Effects of GCV on Tumor-bearing Animals Treated With BacteriaContaining an HSV TK-plasmid

Tumor-bearing mice inoculated with HSV TK plasmid-containing Salmonellaclone YS7211 (YS7211/p5-3) showed suppression of tumor growth andprolonged survival even in the absence of GCV treatment as shown in FIG.11-G. Further, animals bearing both tumors and YS7211/p5-3 andadditionally treated with 3.75 mg GCV showed significant suppression oftumor growth above that seen in the absence of GCV. Using YS7211/p5-3 asa vector, GCV-mediated tumor suppression was evident in adose-responsive manner when measured 28 days post implantation of tumorsas shown in FIG. 11-H. Tumor suppression correlated with increasedaverage survival times for some categories of GCV-treated, tumor-bearingmice when compared to those inoculated with YS7211/p5-3 but notreceiving GCV.

In summary:

1) In tumor-bearing animals not inoculated with Salmonella, GCV had asmall suppressive effect on tumor growth that correlated with a smallprolongation of survival.

2) Tumor-bearing animals inoculated with Salmonella not containing theHSV TK plasmid showed marked tumor suppression in response to GCV, abovethat seen in animals not treated with bacteria. In addition, somecombinations of GCV and bacterial treatment were highly toxic to theanimals, possibly through conversion of GCV to its toxic form bybacteria in extra-tumoral tissues such as liver or bone marrow.

3) Tumor-bearing animals inoculated with Salmonella containing the HSVTK plasmid also showed strong tumor suppression in response to GCV. Itwas not possible in these experiments to evaluate the relativecontributions of ISV TK as compared to endogenous Salmonella enzymes inthe phosphorylation of GCV. However, using as a vector clone YS7211containing the HSV TK expression plasmid, GCV-mediated tumor suppressionand prolonged survival was demonstrated in a dose-dependent manner, seeFIG. 11-H.

17. EXAMPLE Localization of Salmonella typhimurium Within Human TumorsGrown in NU/NU Mice

The following experiments demonstrate localization of Salmonella inhuman tumors in experimental animals.

17.1. Localization of Salmonella Within Human Colon Tumors

NU/NU (BALB C) mice (9-10 weeks old) were inoculated s.c. in two areas(shoulder and flank), each with 1.5×10⁷ HCT 116 human colon carcinomacells. After the appearance of palpable, vascularized tumors(approximately 2 weeks) the animals were further inoculated i.p. with3×10⁵ Salmonella typhimurium super-infective clone 72⁵⁻³⁻² carrying theHSV thymidine kinase gene. After 3.5 hours, 21 hours, and 72 hours ofinfection, mice were euthanized by anesthesia with metofane. Tumors andlivers were removed aseptically, rinsed with sterile NaCl (0.9%),weighed, and homogenized with LB broth at a ration of 5:1 (vol.broth:wt. tumor). At 72 hours, prior to homogenization, pieces (1-2 mm³)were removed from representative tumors, fixed with ½ strengthKarnovsky's fixative, and processed for analysis with the electronmicroscope. Bacteria in the homogenates were quantitated by plating ontoLB plates, incubating overnight at 37° C., and counting bacterialcolonies.

Results were as follows: At 3.5 hours and 21 hours there wereinsignificant levels of bacteria in the tumors or livers, even when thehomogenates were plated undiluted onto LB agar plates. However, after 3days 3/6 animals displayed high levels of Salmonella in the colontumors, with bacterial tumor:liver ratios ranging up to 36,000:1. Datafor these animals are summarized below in Table 20.

TABLE 20 DISTRIBUTION OF SALMONELLA TYPHIMURIUM 3 DAYS FOLLOWINGINOCULATION (I.P.) INTO HUMAN COLON CARCINOMA-BEARING NU/NU MICESalmonella/gm tissue Tumor/Liver Mouse A Liver 2.6 × 10⁴ Tumor 6.9 × 10⁸26,500:1 Mouse D Liver 1.6 × 10⁶ Tumor 3.1 × 10⁹  2,000:1 Mouse E Liver1.0 × 10⁵ Tumor 3.6 × 10⁹ 36,000:1

Shown in FIG. 12-A an electron micrograph of a section from the HCTcolon tumor excised from mouse A (Table 20) in which the number ofSalmonella found to be 6.9×10⁸/g tumor, and the tumor:liver ratio ofinfecting bacteria was 26,500:1. Shown in the micrograph are numerousSalmonella typhimurium within a vacuole in the cytoplasm of a neutrophilassociated with the tumor. Some of the bacteria are undergoing divisionas denoted by the arrow. The neutrophil or polymorphonucleoleukocyte ischaracterized by its multi-lobed nucleus (n). Salmonella intumor-associated neutrophils was also seen in infected B16F10 melanomasas described herein. The presence of bacteria in both colon and melanomatumor-associated neutrophils following infection of tumor-bearing micesuggests that the Salmonella may have stimulated a host cellular immuneresponse to the tumor cells. Enhancement of tumor immunity is thusanother potential advantage in the use of parasites as tumor-specifictherapeutic vectors.

17.2. Localization of Salmonella Within Various Human Tumors

Nu/nu (BALB C) mice (9-12 weeks old) were inoculated s.c. in the leftshoulder region with 1-1.5×10⁷ cells of the human lung carcinoma A549,human colon carcinoma HCT 116, human renal carcinoma CRL 1611, or humanhepatoma HTB 52 (American Type Culture Collection). When palpable tumorsdeveloped, the mice were inoculated further with 2-5×10⁶ cfu Salmonellatyphimurium clone 72 for animals bearing 10 human lung, liver, and renaltumors, and clone 72⁵⁻³⁻² for animals bearing human colon tumors. Clone72⁵⁻³⁻² carries the HSV thymidine kinase transcription unit. After 66-96hours the animals were sacrificed, and the tumors and livers wereremoved and weighed. The tumor was homogenized in 5 vol LB broth/gramwet weight tissue. Homogenates were quantitated by serial dilution on LBagar plates for the number of bacteria. The results are presented inTable 20(A) and represent the average±standard deviation for n=3-4animals.

TABLE 20(A) BIODISTRIBUTION OF SALMONELLA TYPHIMURIUM CLONE 72 IN NU/NUMICE BEARING HUMAN CARCINOMAS OF THE LUNG, COLON, KIDNEY, AND LIVERSalmonella/g tissue: Primary Tumor wt Tumor Tumor Liver (mg) Tumor:Liverlung 3.2 ± 1.4 × 10⁹ 1.0 × 0.3 × 10⁷ 462 ± 186 320:1 carcinoma colon 2.5± 1.6 × 10⁹ 5.8 ± 8.9 × 10⁵ 428 ± 235 4300:1  carcinoma hepatoma 6.7 ±11 × 10⁸ 5.7 ± 9.0 × 10⁶ 103 ± 29  120:1 renal 1.4 ± 1.8 × 10⁸ 6.0 ± 3.0× 10⁵ 103 ± 99  230:1 carcinoma

As shown in Table 20-A, when inoculated i.p. into nu/nu mice, Salmonellatyphimurium clone 72 was able to target human carcinomas of the lung,colon, kidney, and liver, and proliferate within them, generally, butnot always, reaching levels of 10⁸-10⁹/g tumor. In the BALB/c nu/nu miceused, the skin was hairless and translucent allowing it to be determinedvisually that all the tumors were vascularized. The ranges of wetweights of the Salmonella-infected tumors were lung carcinoma, 220-600mg; colon carcinoma, 160-600 mg; hepatoma, 70-120 mg; and renalcarcinoma, 40-250 mg.

Bacterial colonies were picked randomly from liver and tumor homogenatesobtained from renal carcinoma- and hepatoma-bearing nu/nu mice 96 hrspost-inoculation of clone 72 and tested for phenotype by replicateplating. In all homogenates tested, 50/50 colonies were found to beAde⁻and Xyl^(neg), consistent with the clone 72 phenotype.

The results further support the notion that derivatives of Salmonellatyphimurium are useful as therapeutic vectors for a broad range of solidtumors, independent of the tumor origin or size. In several studiesSalmonella clone 72 and its derivatives targeted and amplified withinhighly vascularized tumors as small as 40-100 mg in the case of humantumors in nu/nu mice, as well tumors of 4-8 g with large necrotic areasin the case of B16F10 melanomas in C57B6 mice. The ability to target andamplify within small vascularized tumors presents a distinct advantageof Salmonella typhimurium as a therapeutic tumor vector.

17.3. Localization By Electron Microscopy of Salmonella typhimuriumWithin Human Lung Carcinoma A549

A mouse was inoculated s.c. in the left shoulder region with 5×10⁶ A549cells. After 6 weeks the tumor was palpable and the animal wasinoculated i.p. with 3×10⁶ Salmonella typhimurium clone 72, for 66hours. The animal was sacrificed and a portion of the tumor washomogenized and found to contain 1.6×10⁹ Salmonella typhimurium/g. Thecentral portion of the tumor was prepared for electron microscopy asfollows: The portion of the tumor was cut into 1-2 mm³ pieces and fixedin ½ strength Karnovsky's fixative for 6 hours at 4° C., followed bywashing in cacodylate buffer overnight. The tumor tissue was post-fixedwith 1% OsO₄ and 1.5% potassium ferrocyanide in cacodylate buffer for 2hours and embedded in Spurr's resin. Ultrathin sections were stainedwith uranyl acetate and lead citrate. They were photographed through aZeiss 109 electron microscope. It should be noted for comparisonpurposes that the S. typhimurium shown in the electron micrograph ofFIG. 12-B appear similar to those shown previously in intestinalepithelial cells following an experimental infection of the mouse,Takeuchi, 1967, Am. J. Pathol. 50:109-1361.

Shown in FIG. 12-B are numerous Salmonella typhimurium, denoted byarrows, in extracellular spaces as well as contained within a singlecell, possibly a neutrophil, seen in the upper left. Also seen in thefield are two unidentified cells that appear to be dying as indicated bythe large intracellular space, along with cellular debris.

18. EXAMPLE Attenuation of Salmonella typhimurium By Mutation toAuxotrophy

The studies below demonstrate that the reduced virulence of clone 72(see, e.g., Section 15.2 above) is due to a Pur⁻phenotype. Furtherdescribed are analyses of a virulent derivatives of clone 72 that wereisolated as additional auxotrophic mutants, expressing in differentcombinations the phenotypes of Ade⁻, Ilv⁻, Arg⁻, Aro⁻, and Ura⁻.

18.1. Mutation to Auxotrophy

Clone 72 was examined for auxotrophic mutations and was found to havegrowth requirements for both adenine and vitamin B1, indicating amutation(s) in the purine biosynthetic pathway (Pur⁻). An experiment wasdesigned to test whether the ade⁻ mutation could account for theobserved attenuation of clone 72 described above. Populations of bothwild type strain 14028 and clone 72 were mutagenized with UV radiationand nitrosoguanidine as described in Section 7.1. From the population ofmutagenized strain 14028, three separate Pur⁻ auxotrophic mutant cloneswere isolated and designated clones N, Q, and T. From the population ofmutagenized clone 72, three separate Pur⁺ revertant clones were isolatedand designated clones R, U, and W.

C57B/6 mice were injected i.p. with 2×10⁶ c.f.u. Salmonella typhimuriumof each of the strains obtained. The mice were allowed to eat and drinkad libitum and the cages were monitored for dead or moribund mice.Moribund animals (listless, cessation of drinking) were euthanized andcounted with the other dead. After 10 or 30 days post-injection withbacteria the surviving animals were euthanized.

The results are shown in Table 20(B).

TABLE 20(B) SURVIVAL OF C57B6 MICE INJECTED WITH DIFFERENT AUXOTROPHICMUTANTS OF SALMONELLA TYPHIMURIUM Time of Death Survivors SurvivorsStrain Phenotype (Days ± S.D.) >10 days >30 days 14028 wild type 3.0 ±0.5 n.a. n.a. 72 superinfective, ade⁻ 5.8 ± 1.4 n.a. n.a. R 72, Pur⁺ 3.9± 0.4 n.a. n.a. U 72, Pur⁺ 3.9 ± 1.3 n.a. n.a. W 72, Pur⁺ 4.1 ± 0.9 n.a.n.a. T 14028, Pur⁻ 6.8 ± 1.5 n.a. n.a. N 14028, Pur⁻ n.a. 4/8 n.d. Q14028, Pur⁻ n.a. 5/8 n.d. YS721 72, Ilv⁻ n.a. 10/11  6/11 YS7211 72,Ilv⁻, Arg⁻ n.a. 8/8 7/8 YS7213 72, Ilv⁻, Aro⁻ n.a. 8/8 8/8 YS7212 72,Ilv⁻, Ura⁻ n.a. 8/8 6/8 Results are the average ± SD for n = 8-12animals n.a., not applicable; n.d., not done.

As shown in Table 20(B), Clone 72 was less virulent than the wild typestrain 14028. However, 3 of 3 Pur⁺ revertants of clone 72 (U, W, and T)expressed virulence similar to 14028. Conversely, 3 of 3 Pur⁻auxotrophic mutants isolated from strain 14028 (T, N, and Q) were lessvirulent than either 14028 or clone 72.

Isolation of additional auxotrophs from clone 72 produced even lessvirulent strains. For example, clone YS721 is an isoleucine-valinerequiring (Ilv⁻) derivative of clone 72, and clone YS721 wassignificantly less virulent than clone 72. Similarly, auxotrophicderivatives of clone YS721 such as clones YS7211 (Arg⁻) , YS7212 (Ura⁻),and YS7213 (Aro⁻) were all significantly less virulent than YS721itself.

18.2. Evidence that the Superinfective Phenotype of Clone 72 IsGenetically Distinct From its Auxotrophic Purine Requirement

The various Salmonella typhimurium Pur⁻ and Pur⁺ strains described abovein Section 18.1 were assayed for their ability to infect human M2melanoma cells in culture. The in vitro infection assay employed was asdescribed in Section 18.1.

The results are described in Table 20(C).

TABLE 20(C) INFECTIVITY TOWARD HUMAN M2 MELANOMA CELL IN VITRO BYVARIOUS PURINE MUTANTS OF SALMONELLA TYPHIMURIUM Infecting Salmonella/10⁶ melanoma Strain Phenotype cells/15′ (±S.D.) ×wild type 14028 wildtype 1.0 ± 0.2 × 10⁵ 1.0× 72 superinfective, ade⁻ 9.8 ± 0.7 × 10⁵ 9.8× R72, Pur⁺ 5.9 ± 1.4 × 10⁵ 5.9× U 72, Pur⁺ 1.1 ± 0.2 × 10⁶  11× W 72, Pur⁺1.1 ± 0.3 × 10⁶  11× N 14028, Pur⁻ 1.9 ± 0.5 × 10⁵ 1.9× Q 14028, Pur⁻1.5 ± 1.0 × 10⁵ 1.5× T 14028, Pur⁻ 1.1 ± 0.4 × 10⁵ 1.5× Results are theaverage ± SD for triplicate infections. The bacteria were cultured in LBbroth to O.D.₆₀₀ = .600 prior to their dilution and use in the infectionassays.

As shown in Table 20(C), Clone 72 displayed superinfectivity towardhuman M2 melanoma cells compared to wild type strain 14028. None of the14028 Pur⁻ derivatives differed significantly in its infectivity fromstrain 14028 itself, and all of the clone 72 Pur⁺ derivatives expressedsuperinfectivity similar to clone 72 itself. The results demonstratethat the purine requirement exhibited by clone 72 which accounts for thereduced virulence of clone 72 in mice, is genetically separate from thesuperinfective phenotype of clone 72. These results demonstrate thatneither mutation to nor reversion from purine auxotrophy effectsexpression of the superinfective phenotype characteristic of clone 72.

18.3. Retention of the Superinfective Phenotype By AttenuatedDerivatives of Salmonella typhimurium Clone 72

In the experiments below, the infectivity of certain auxotrophicderivatives of clone 72 described above in Section 18.1 was assessed invitro. The phenotypes of the clones of Salmonella evaluated are shown inTable 20(B) in Section 18.1 above. Infectivity assays described inSection 10.1 were employed.

The results are presented in Table 20(D).

TABLE 20(D) INFECTIVITY OF SALMONELLA TYPHIMURIUM AUXOTROPHS TOWARDHUMAN MELANOMA CELLS IN CULTURE Infecting Salmonella/ Strain 10⁶melanoma cells/15 min ×wild type 14028 (wild type) 4.3± × 10⁴ 1.0× clone72 4.4± × 10⁵  10× clone YS721 3.2± × 10⁵ 7.4× clone YS7211 2.0± × 10⁵4.7× clone YS7212 1.7± × 10⁵ 4.0× clone YS7213 1.3± × 10³ 0.03×  Theresults represent the average ± SD for 10-19 separate infections. Thebacteria were grown in LB broth to O.D.₆₀₀ = 0.6 before being dilutedprior to their use in the infection assays

Salmonella typhimurium clones YS721, YS7211, and YS7212, though eachsomewhat less infective of M2 melanoma cells than clone 72, werenonetheless superinfective when compared to wild type strain 14028,indicating their partial retention of the superinfective phenotype. Incontrast, clone YS7213 (Ade⁻, Ilv⁻, Aro⁻) was found to have greatlyreduced infectivity, being about 30-fold less infectious toward M2melanoma cells than the wild type strain 14028.

18.4. Growth of Pur⁻ and Ura⁻ Mutants of Salmonella typhimurium WithNutritional Additives or Extracts of B16F10 Melanoma

Tumor extracts were prepared in the following manner: B16F10 melanomatumor cells (5×10⁵) were implanted s.c. into 68 week female C57B6 mice.After 3-4 weeks, the mice were sacrificed and the tumors removedaseptically and rapidly frozen, −20° C. A total of 51 g of frozen pooledtumors was thawed at 4° C. and vigorously homogenized in 255 ml (5 vol)H₂O in a capped Virtis tissue homogenizer in the cold for 1 hour. Theresulting homogenate was made lot with trichloracetic acid (TCA), placedon ice for 15 minutes, and centrifuged in a Beckman J21 centrifuge atabout 20,000×g for 15 minutes at 4° C. Further procedures were conducedat room temperature. The clear, colorless supernatant fraction (300 ml)was retained and extracted by manual shaking for 1 minute with 1 volume(300 ml) anhydrous ether. Between extractions, the mixtures were allowedto settle and the upper phase (containing ether, extracted TCA, as wellas ether-soluble compounds from the tumor extract) was removed byaspiration and discarded through approved environmentally-protectiveprocedures. During 5 such extraction cycles, the pH of the water phaserose from a starting value of about pH 1 to a final value of pH 4-5,similar to that of distilled H₂O, indicating that the TCA had beeneffectively removed. The water phase was bubbled with a stream ofnitrogen for about 15 minutes, at which time the odor of ether haddisappeared.

The solution was then filtered through a 0.2 micron filter, divided intoaliquots and either used directly in the assays herein, or stored at−20° C. for further use.

Wild type strain 14028, and its auxotrophic derivatives clone 72 (Pur⁻,vitamin B1⁻), and YS7212 (Ade⁻, vitamin B1⁻, Ilv⁻, Ura⁻) were grownovernight on a slant in 5 ml Luria broth (LB) at 35° C. The next day 0.1ml of each culture was diluted into 10 ml of Medium 56 (0.037 M KH₂PO₄,0.06 M Na₂HPO₄, 0.020% MgSO₄-7H₂O, 0.2% (NH₄)₂SO₄, 0.001% Ca(NO₃)₂ and0.00005% FeSO₄-7H₂O) supplemented with 0.2 μg/ml vitamin B1, 33 μg/mladenine, 50 μg/ml uracil, 83 μg/ml isoleucine, 83 μg/ml valine and 0.3%glucose, and grown on a rotor overnight at 37° C. The next day thecultures were collected by centrifugation and resuspended in plainMedium 56 (1 ml culture plus 9 ml Medium 56). Aliquots (0.25 ml) ofthese suspensions were then added to Medium 56 containing varioussupplements in the following manner:

A. Medium 56 plus glucose;

B. Medium 56 plus glucose, vitamin B1, adenine, isoleucine, valine, anduracil; and

C. Medium 56 and tumor extract (10%).

The bacteria were placed in a swirling H₂O bath, 37° C., and growth as afunction of OD₆₀₀ was followed with a spectrophotometer. The startingoptical densities for all of the cultures ranged from 0.005-0.07.

As demonstrated in FIGS. 15A-C, wild type strain 14028 was able toproliferate at about the same rates in all three of the media tested,including the most basic of the three, Medium 56 plus glucose. Unlikethe wild type strain, neither clone 72 nor clone YS7212 was able to growin Medium 56 plus glucose, indicative of their nutritional requirementsoriginally observed through replicated plating on agar. In contrast bothclone 72 and clone YS7212 were able to grow in Medium 56 supplementedwith 10% tumor extract. Liver extracts prepared in the same manner werealso able to support the growth of clones 72 and YS7212.

Although the inventors do not wish to be limited to a specific mechanismof action, since the growth state of auxotrophic strains of Salmonellais dependent upon the availability of nutrients, such auxotrophs wouldseem to have advantages as tumor vectors since the environment of thetumor could in theory provide such nutrients, for example in necroticspaces or within actively dividing cells of the tumor. Thus, mutation oforganisms such as Salmonella to auxotrophy not only reduces theirvirulence in vivo but also may provide a potential mechanism for theirselective population and amplification within solid tumors.

18.5. Proliferation of Pur⁻ and Ura⁻ Mutants of Salmonella typhimuriumin Human M2 Melanoma Cells in Culture

In this Section it is demonstrated that the internal milieu of culturedM2 melanoma cells also can also supply the auxotrophic requirements ofthese clones, since both clone 72 and clone YS7212 were able to undergoseveral rounds of division once they invaded M2 melanoma cells culturedunder aerobic conditions.

Salmonella typhimurium clones 72 and YS7212 were grown to O.D.₆₀₀=0.8,or about 10⁹ c.f.u./ml. The two strains were then added at 10⁶c.f.u./mlculture media of human M2 melanoma cells as described above in Section7.2. 15 minutes after infection with Salmonella, the eukaryotic cellcultures were rinsed with fresh medium and medium containing gentamicin(10 μg/ml) was added. At hourly intervals over a 6 hour period, cultureswere processed as described in Section 7.2 for quantitation ofSalmonella/10⁶ melanoma cells. In addition, control flasks withoutmelanoma cells but with bacteria were processed side-by-side with theexperimental flasks containing melanoma cells.

The results are shown in FIG. 15-D. Control flasks with Salmonella butwithout melanoma cells showed no viable bacteria over the 6 hour period,demonstrating that the wash procedure coupled with gentamicin treatmentsuccessfully eliminated all viable bacteria not protected by locationwithin animal cells. In contrast, in the presence of M2 melanoma cells,Salmonella typhimurium clones 72 and YS7212 each increased significantlyin number over the 6 hour period with doubling times of about 2 hoursfor each strain. Phase and electron microscope analyses demonstratedthat M2 melanoma cells were able to compartmentalize infectingSalmonella within vacuoles. The results indicate that the net rate ofgrowth of Salmonella within the melanoma cells was a steady-statefunction, reflecting the ability of the melanoma cells to both stimulategrowth of the auxotrophs through the supply of nutritional requirements,as well as to suppress the growth of the auxotrophs throughanti-bacterial mechanisms.

18.6. Biodistribution of Autotrophic Attenuated Strains of Salmonellatyphimurium in C57B6 Mice-Bearing B16 Melanoma Tumors

These studies demonstrate the ability of clones YS721, YS7213, YS7211and YS7212 to target tumors and proliferate within the tumor in vivo.

C57B6 6-8 week old female mice were inoculated s.c. (left flank) with2.5-5.0×10⁵ B16F10 mouse melanoma cells. When the tumors reached about0.5 g (14-16 days post-tumor inoculum), the animals were furtherinoculated i.p. with the indicated strains of S. typhimurium. Thebacterial inoculum was 4×10⁵ cfu/mouse for strains 14028 and 72 and2-4×10⁶ cfu/mouse for strains YS721, YS7211, YS7212 and YS7213. After 40and 96 hours post-inoculation of bacteria, the mice were sacrificed, thetumors and livers were removed aseptically, rinsed with sterile NaCl(0.9%), weighed, and homogenized in LB broth at a ratio of 5:1(vol:tumor wt). Bacteria were quantitated by plating the homogenatesonto LB plates, incubating overnight at 37° C., and counting bacterialcolonies. The results presented in Table 20(E) represent the average±SDfor n=4-7 animals.

TABLE 20(E) BIODISTRIBUTION OF WILD TYPE AND ATTENUATED STRAINS OFSALMONELLA TYPHIMURIUM IN C57B6 MICE-BEARING B16 MELANOMA TUMORSSalmonella/g tissue: Strain Tumor Liver Tumor:Liver A. 40 hrspost-inoculation of bacteria 14028 6.5 ± 6.8 × 10⁹ 2.4 ± 2.8 × 10⁷ 270:172 1.7 ± 1.2 × 10⁹ 1.9 ± 2.3 × 10⁵ 9000:1  YS721 8.7 ± 3.1 × 10⁸ 4.2 ±3.6 × 10⁶ 210:1 YS7211 3.3 ± 3.0 × 10⁷ 8.1 ± 8.4 × 10⁵  41:1 YS7212 3.9± 7.3 × 10⁷ 1.1 ± 0.8 × 10⁶  35:1 YS7213 1.5 ± 2.8 × 10⁸ 4.0 ± 3.1 × 10⁵375:1 B. 96 hrs post-inocu1ation with bacteria 14028 moribund/dead 72moribund/dead YS721 3.2 ± 1.5 × 10⁹ 4.7 ± 6.9 × 10⁶ 680:1 YS7211 1.6 ±2.2 × 10⁹ 6.3 ± 9.9 × 10⁶ 253:1 YS7212 1.1 ± 7.4 × 10⁹ 5.1 ± 8.6 × 10⁵2200:1  YS7213 1.3 ± 2.5 × 10⁹ 2.2 ± 6.9 × 10⁵ 5900:1 

Each of the strains tested was able to target the tumor tissue andreplicate to varying degrees within the tumor, as evidenced by thefinding that in all cases the tumors contained 10-1000 times moreSalmonella typhimurium than were first inoculated. Further, in all casesthe tumor:liver ratio of bacteria/g tissue was at least 35:1 and in somecases approached 10⁴. The tumors analyzed in the studies presented inTable 20(E) ranged in weights from 0.5-2.0 g. Of all the conditions andstrains tested, clone 72 exhibited the highest tumor:liver ratio 40hours post inoculation. Further, Salmonella typhimurium, strain 14028,as well as clone 72 and its derivatives were also able to target andamplify within larger B16P10 melanoma tumors of, for example, 4-8 g. Inaddition, as shown in Section 10.3.2 and Table 12A, clone 72 can targetand amplify within human solid tumors as small as 40 mg.

However, both clone 72 and the wild type strain 14028 were highlyvirulent toward C57B6 mice, especially mice bearing tumors. For example,C57B/6 mice bearing B16F10 melanomas injected with strains 1428 and 72had average survival times of 2.1±0.4 days (n=6) and 4.7±0.5 days (n=9)post-inoculation of bacteria respectively. The biodistribution of thesestrains was thus not measured at 96 hours. Likewise, clone YS721, thoughattenuated compared to 14028 and clone 72, was nonetheless virulent inmelanoma-bearing mice. For example, B16F10 melanoma-bearing C57B6 miceinjected with clone YS721 had an average survival time of 8.1±0.2 days(n=11). Salmonella clones YS7211, YS7212 and YS7213, the least virulentof those examined, each displayed densities of greater than 10⁹ cfubacteria/g tumor 96 hrs post-inoculation with tumor:liver ratios of253:1, 2200:1, and 5900:1 respectively.

18.7. Phenotypic Stability Following Incubation of Salmonellatyphimurium Auxotrophs in Tumor-Bearing Mice

Genetic reversion of an auxotrophic phenotype could in theory result inan increase in virulence of the previously attenuated bacteria.Therefore, the stabilities of the auxotrophic phenotypes of the strainsYS7211, YS7212 and YS7213 were tested following incubation of thebacteria in tumor-bearing mice.

Salmonella typhimurium obtained from the homogenates of livers andtumors of animals 40 hours post-inoculation of either YS7211, YS7212 orYS7213 were picked from LB plates and replicate plated onto minimalmedia agar plates supplemented with nutritional additives in differentcombinations. The supplements were isoleucine, valine, adenine/vitaminB1, arginine, uracil, aromatic amino acids, and glucose. For each of thethree strains, 50/50 of the bacterial clones recovered from the tumorand liver homogenates displayed the expected phenotypes of the strainoriginally inoculated, indicating that in this experiment the strainswere genetically stable enough not to revert substantially under theconditions tested.

However, it should be noted that the auxotrophic strains employed werenot absolutely stable throughout these studies. In some cases geneticrevertants were observed, most notably in the YS7211 strain whereinrevertants from Arg⁻ to Arg⁺ were observed. For example, in atumor-bearing mouse inoculated 96 hours earlier with clone YS7211bearing a thymidine kinase-containing plasmid, 50 out of 50 bacteriaisolated from the liver were found to be Pur⁻, Ilv⁻ and Arg⁺, indicatingthat reversion and selective growth of the reverted organisms hadoccurred within the mouse. The finding that the auxotrophic phenotypesof clones YS7211, YS7212 and YS7213 were relatively stable in mice wassupported by the long term survival of mice inoculated with thesestrains.

18.8. Suppression of Tumor Growth and Increased Survival of C57B6Tumor-Bearing Mice Inoculated With Auxotrophic Mutants of Salmonellatyphimurium

C57B6 female mice, 5-7 weeks old, were inoculated s.c. in the leftshoulder region with 5×10⁵ B16F10 melanoma cells grown in culture. Onthe 8th day following inoculation of tumor cells, the mice were furtherinoculated i.p. with 2-4×10⁶ c.f.u. of Salmonella typhimurium strainsYS721, YS7211, YS7212 or YS7213. Tumor growth was assessed with periodiccaliper measurements of tumor length, width and height, and computed astumor volume in mm³. Results of tumor growth, shown in FIGS. 16A-Drepresent the averages±SD for 5 animals/group with 5/5 animalssurviving. After the point at which one or more animals died within agroup, the average tumor sizes of the surviving animals were no longershown when the data were plotted as shown in FIGS. 16A-D.

All tumor measurements were stopped after 33 days post implantation oftumor cells, even though 5/5: tumor-bearing animals treated with cloneYS7211 were still alive at this time. The animals were allowed to eatand drink ad libitum. Twenty-three days (Experiment #1) or 10 days(Experiment #2) following inoculation of bacteria, both control andbacteria treated mice were given Baytril™ (enrofloxacin, 0.2 mg/mldrinking water) and maintained on this antibiotic for a total of 2weeks. In Experiment #1 the times at which the mice became moribund(listless, cessation of drinking) or died, were noted. The results arepresented in Table 20(F) as the average survival±SD for the conditionstested. In Experiment #2 animals were sacrificed when the tumor reached4 g and listed with the other dead as described in Experiment #1. Thetwo different methods for assessing survival accounted for a somewhatshorter survival time for control animals in Experiment #2 (26 days) ascompared to Experiment #1 (28 days).

TABLE 20(F) SURVIVAL OF B16F10 MELANOMA-BEARING C57B6 MICE INOCULATEDWITH SALMONELLA TYPHIMURIUM Time of death post tumor cell inoculation:Strain (Days ± SD) Treated/Control (T/C) Control (no bacteria) Expt 1 28± 2 1.0 2 26 ± 3 1.0 YS7211 1 36 ± 9 1.3 2  41 ± 10 1.6 YS7213 1 36 ± 51.3 2 38 ± 6 1.5 YS7212 1 51 ± 7 1.8 2 55 ± 3 2.1 The results representthe average ± SD for 6 animals.

FIGS. 16A-D shows the average±SD tumor volumes (mm³) versus time postinoculation of 5×10⁵B16F10 melanoma cells s.c. into C57B/6 mice. Allfour clones of Salmonella, namely clones YS721, YS7211, YS7212 andYS7213, elicited suppression of tumor growth in the animals. CloneYS721, attenuated through Ade⁻ and Ilv⁻ auxotrophy, was nonethelesstoxic to tumor-bearing mice and resulted in no prolongation of survivalcompared to control tumor-bearing animals receiving no bacteria. Whereasthe death of control animals was clearly due to very large tumor masses(4-8 g), the death of tumor-bearing animals inoculated with clone YS721appeared to be a result of bacterial toxicity since the tumor burden inthese animals was quite small and not life-threatening in itself. Thetumors ranged from non-palpable to less than 0.5 g at the time of death.

In contrast, treatment of tumor-bearing mice with clone YS7211, YS7212and YS7213, each less virulent than clone YS721, resulted in significantenhancement of survival in addition to suppression of tumor growth. Thedegree of suppression of tumor growth by the individual Salmonellaclones, as seen in FIGS. 16A-D, correlated with their abilities toelicit enhanced survival, as seen in Table 20(F). The average time fortumors to reach 1 g (1000 mm³) was about 18 days for control animals, 31days for animals treated either with YS7213 and YS7211 and 45 days(extrapolated) for YS7212. This corresponded to average survival timesfor 26 days for control tumor-bearing animals, compared to 38, 41, and55 days for animals treated with clones YS7213, YS7211 and YS7212.

Thus, among the attenuated strain of Salmonella tested, the order ofefficacy for suppression of tumor growth and prolongation of survivalwas YS7212>YS7211>YS7213. Earlier treatment with an antibiotic,enrofloxacin, i.e., 10 days as compared to 23 days post-inoculation ofbacteria, increased the survival time for tumor-bearing animalsinoculated with Salmonella, but not that of control animals.

18.9. Anti-tumor Activity of Auxotrophic Salmonella typhimuriumExpressing Cytosine Deaminase

Experimental metastasis model of B16F10was set up by injecting 1×10⁵cells into C57B/6 mice via the lateral tail vein on Day 0. Aliquots of0.2 ml bacterial suspension of YS7212 carrying the cytosine deaminaseexpression construct (see FIG. 4E for the CD construct) (approximately1×10⁷ CFU/ml) were injected intraperitoneally into mice on Day 5.5-Flourocytosine (5-FC), at 0.4 ml aliquots dissolved in PBS at 10 mg/ml(final dose: 200 mg/kg), was injected into mice intraperitoneally on Day7. Death of animals was recorded daily. Results are presented in FIG.17.

FIG. 17 clearly demonstrates that combination of CD and 5-fluorocytosineprolong the survival Salmonella expressing animals bearing B16F10 lungmetastases.

19. EXAMPLE Attenuation of Salmonella typhimurium Through Mutation inLipoplysaccharide Biosynthesis

Several mutant strains of Salmonella typhimurium and E. coli have beenisolated with genetic and enzymatic lesions in the LPS pathway (Raetz,1993, J. Bacteriol. 175:5745-5753). One such mutant, the firA⁻ mutationis within the gene that encodes the enzyme UDP-3-O(R-30hydroxymyristoyl)-glycocyamine N-acyltransferase, that regulates thethird step in endotoxin biosynthesis (Kelley et al., 1993, J. Biol.Chem. 268:19866-19874). Salmonella typhimurium and E. coli strainsbearing this type of mutation produce a lipid A that differs from wildtype lipid A in that it contains a seventh fatty acid, a hexadecanoicacid (Roy and Coleman, 1994, J. Bacteriol. 176:1639-1646) and hasdecreased lipid A 4′ kinase activity.

A firA⁻mutant was investigated for its ability to induce TNFα productionby human monocytes as well as its ability to target solid tumors inmice.

19.1. Ability of Salmonella typhimurium firA⁻ to Induce TNF-α ProductionBy Human Blood Monocytes

Salmonella typhimurium strain SH5014 and its firA⁻ derivative SH7622 aredescribed in Hirvas et al., 1991, EMBO J. 10:1017-1023. The genotypes ofthese strains are as follows:

strain SH5014 ilv-1178 thr-914 his-6116 metA22 metE551 trpB2 xyl-404H1-b H2-e,n,x flaA66 rpsL120 rfaJ4041;

strain SH7622 ilv-1178 thr-914 his-6116 metA22 metE551 trpB2 xyl-404H1-b H2-e,n,x flaA66 rpsL120 rfaJ4041, ssc-1(firA^(ts)).

A derivative of Salmonella typhimurium firA⁻ strain SH7622 was picked,designated SH7622-64, and used as the firA⁻ strain for the experimentsin this section as well as in Section 19.2 below. SH7622-64 was selectedfor its supersensitivity to the antibiotic novobiocin andtemperature-sensitive growth, characteristics of the firA⁻ SH7622strain.

LPS was extracted from Salmonella typhimurium strain 14028 and itsderivatives clone 72, clone YS7212, and clone YS7213; as well as strainSH5014 and its firA⁻ derivative, clone SH7622-64, as follows: Thebacteria were grown in 500 ml LB broth to O.D.₆₀₀=0.9 or about 2×10⁹cfu/ml. They were collected by centrifugation, and the pellets,containing about 10⁹ cfu/ml. They were collected by centrifugation, andthe pellets, containing 10¹² bacteria, were drained and stored frozen at−20° C. To extract LPS, the pellets were resuspended in 18.3 ml H₂O, and15 ml redistilled phenol was added (H₂O:phenol, 55:45, vol/vol). Themixtures were placed in a shaking water bath at 69-70° C., for 1 hourproducing a monophasic mixture, and then cooled on ice. On cooling themixture separated into a phenol phase containing mainly proteins, and awater phase containing lipopolysaccharide and nucleic acid (Galanos, C.,Luderitz, O., and Westphal, O., 1969). The water phase was lyophilizedto dryness and the white fluffy lyophilized material was used as thesource of LPS. The LPS was weighed and dissolved in H₂O at 1 mg/ml, asstock for dilution in the incubations with human macrophages describedbelow.

Human macrophages were prepared as follows and all procedures were atroom temperature: Blood (60 ml) was collected from a healthy humanvolunteer into a heparinized syringe. The blood was layered in 7 mlaliquots over 4 ml of Isolymph™ (density—1.077 g/ml; 9.0 g sodiumdiatrizoate and 5.7 g Ficoll 400™/100 ml H₂O; Pharmacia Fine Chemicals,A.B. Uppsala, Sweden) in 15 ml Corning Plastic Centrifuge tubes,centrifuged at 2000×g for 45 minutes. The red blood cells pelletedthrough the Isolymph™, neutrophils and other cells sedimented in adiscrete band above this interface, and above the lymphocyte/macrophageband was serum, visible by its yellow color. The serum from each tubewas removed by pipette, pooled in a total volume of about 30 ml andsaved for supplementation into the culture media as described below. Thelymphocyte/macrophage bands were pooled in a total volume of about 15 mldiluted with 40 ml RPMI 1640 culture medium, and centrifuged at 1000×gfor 5 minutes. The cloudy supernatant was discarded and about 0.2 ml ofpelleted white cells was obtained. The cells were resuspended with 50 mlRPMI 1640 culture medium supplemented with 15% human serum (describedabove), penicillin (100 units/ml) and streptomycin (100 μg/ml). Therecovery of viable lymphocytes and macrophages from 60 ml whole bloodwas determined by hemocytometer counting to be about 7×10⁷ cells.Together the cells, lymphocytes and monocytes were distributed into 24well Corning Tissue Culture Plates at 0.5 ml/well, and incubated in agassed humidified incubator at 37° C. for 15 hours.

The next day the cultures were rinsed twice with serum-free RPMI 1640containing antibiotics. Between each rinse, the cultures were incubatedabout 1 hour in the 37° C. incubator to facilitate removal oflymphocytes and other non-adherent cells. Adherent cells were found tobe mostly, if not all derived from blood monocytes, i.e., macrophagesthat had differentiated from their blood monocyte state by virtue ofattachment to the culture dish. For example, in a histochemical assay todetermine the percentage of macrophages in the adherent population ofcells 48 hours post-plating into culture, the population was found to beessentially 100% positive for expression of the enzyme non-specificesterase, a marker commonly used to distinguish monocytes andmacrophages from lymphocytes and other cell types. Results indicatedthat most if not all of the cells employed in the LPS challengedescribed below were of monocyte origin.

After the second rinse described above, serum-free, antibioticcontaining RPMI 1640 supplemented with LPS at the concentrationsindicated was added to the cells, and the cultures were placed in agassed, humidified incubator at 37° C. overnight. After 20 hours, thewell plate cultures were centrifuged in a Beckman GS-15 centrifuge at8000×g for 10 minutes, and the supernatants were removed and assayed forTNF-α content using the QUANTIKINE™ Human TNF-α Immunoassay Kit #DTA50(R&D Systems, Minneapolis, Minn.). TNF-α production as pg/ml by humanmacrophages is plotted as a function of pg/ml bacterial LPS added to theculture medium and shown in FIG. 18.

Strain 14028 and its derivatives clone 72, clone YS7212, and cloneYS7213, as well as strain SH5014, all induced TNF-α production by humanmacrophages at concentrations of LPS in a dose-dependent fashion.Concentrations of LPS from each of these strains as low as 100 pg/ml(0.1 ng/ml) were stimulatory to TNF-α production, and increasinglystimulatory at concentrations of 10³ pg/ml and 10⁴ pg/ml, inducing TNF-αproduction by macrophages to levels of 600-800 pg/ml. The levels ofTNF-α induced by the LPS were similar to the circulating levels of TNF-αfound in patients with septic shock syndrome as well as in humanvolunteers injected with E. coli LPS (Morrison et al., 1994, ASM News60:479-484). In contrast, LPS from firA⁻strain SH7622-64 was far lessstimulatory to TNF-α production by the macrophages, and was detectedonly at concentrations of 10⁴ pg/ml. Thus, on a dose responsecomparison, LPS from strain SH7622-64 was only about 1% as effective instimulating macrophage TNF-α production when compared to LPS from thefirA⁺ parental strain SH5014. Furthermore, the results demonstrate thatstrains YS7212 and YS7213 each produced LPS similar to wild type strain14028 LPS, as assessed by stimulation of human macrophages.

19.2. Tumor Targeting By Salmonella typhimurium Bearing the firA⁻Mutation

M27 mouse lung tumor cells or B16F10 mouse melanoma cells (5×10⁵) wereimplanted s.c. in C57B6 mice. When the tumors were palpable, SH7622-64grown in LB broth at 37° C. to a density of about 10⁹ cfu/ml(OD₆₀₀=0.8). Aliquots of 5-10×10⁶ cfu were removed and inoculated intotumor bearing mice. At 48 hrs (M27 lung) and 96 hrs (B16F10 melanoma)post-inoculation of bacteria the animals were sacrificed, the tumors andlivers removed, weighed and homogenized in LB broth at a ratio of 5 ml/gtissue. Homogenates were quantitated for bacteria by serial dilutions onLB agar plates. Results are presented in Table 20(G).

TABLE 20(G) TUMOR LOCATION BY SALMONELLA TYPHIMURIUM BEARING THE firA⁻MUTATION FOR LIPOPOLYSACCHARIDE BIOSYNTHESIS Salmonella/g tissue:Primary Tumor Tumor Liver Tumor:Liver M27 lung 2.9 × 10⁶ -0- n.a. B163.2 × 10⁶ 1 × 10² 3200:1 The results are derived from single anlmals.

As shown in Table 20(G), strain SH7622-64 was able to locate within boththe B16F10 melanoma and the M27 lung tumors when inoculated i.p. intomice. These results, in combination with those in Section 19.1 whichshow that LPS from this particular firA⁻ strain was greatly suppressedin its ability to induce TNF-α in human macrophages, demonstrate thatSalmonella attenuated through a mutation in endotoxin biosynthesis canbe useful as tumor vectors in vivo.

20. EXAMPLE Tumor-Specific Accumulation of Clones YS721 and YS7211 inMurine Lewis Lung Carcinoma

This example demonstrates that auxotrophic mutant Salmonella clonesYS721 and YS7211 locate to lung carcinoma.

The experimental model of the Lewis lung carcinoma was set up byinjecting 5×10⁵ cells into C57B/6 mice subcutaneously on Day 0. Aliquotsof 0.2 ml bacterial suspension (approximately 1×10⁷ CFU/ml) wereinjected intraperitoneally into mice on Day 14. On Day 16, the tumorsand livers were harvested and homogenized and bacterial countsdetermined by plating serial dilutions. Results of the relativedistribution are shown in Table 20(H).

TABLE 20(H) TUMOR SPECIFIC ACCUMULATION OF CLONES YS721 and YS721 1 INMICE No. pathogens/ No. pathogens/ Strain g Liver g TumorRatio:tumor/liver YS721 9.8 × 10⁶ 4.7 × 10¹⁰ 4.8 × 10³ 4.3 × 10⁵ 3.2 ×10¹⁰ 7.3 × 10⁴ 1.1 × 10⁶ 1.4 × 10⁹  1.3 × 10³ 1.6 × 10⁶ 1.0 × 10¹² 6.2 ×10⁶ 3.0 × 10⁴ 2.3 × 10⁹  7.7 × 10⁴ YS7211 1.4 × 10⁴ 2.6 × 10¹⁰ 1.9 × 10⁶1.9 × 10⁵ 2.7 × 10⁶  1.4 × 10³ 2.3 × 10⁵ 6.0 × 10¹¹ 2.6 × 10⁶ 1.0 × 10⁶5.0 × 10¹¹ 5.0 × 10⁵

Extremely high levels of bacteria were localized to these tumors, aswell as others indicating that the auxotrophic mutations retain tumorspecific accumulation of bacteria for a spectrum of tumor models.

21. EXAMPLE Treatment of B16F10 Melanoma Metastatic Tumors

Metastases constitute one of the major problems for treatment of solidtumors. While larger tumors can be detected and removed surgically,smaller metastases. constitute the untreated reservoir which isfrequently the cause of death. Therefore, an effective cancertherapeutic should be effective against metastatic tumors.

An experimental metastasis model of B16F10 was set up by injecting 1×10⁵CELLS into C57B/6 mice via the lateral tail vein on Day 0. Aliquots of0.2 ml YS7211/p5-3 and YS7212/p5-3 (YS7211 and YS7212 each carrying theHSV thymidine kinase expression plasmid) bacterial suspensions(approximately 1×10⁷ CFU/ml) were injected intraperitoneally into miceon Day 5. Ganciclovir, at 0.1 ML aliquots dissolved in PBS at 22 mg/ml(final dose: 100 mg/kg), was injected into mice intraperitoneally on Day7. Tumor progression was monitored by periodic sacrifice and examinationof the lungs. At day 28, all the animals were sacrificed and the normaland tumor-bearing lungs weighted.

FIG. 19 clearly demonstrates that animals inoculated with YS7212carrying the HSV thymidine kinase gene (YS7212/p5-3) and further treatedwith GCV show reductions in the number and extent of B16F10 lungmetastases.

22. EXAMPLE Diagnosis of Tissue Biopsies For Melanoma Using Salmonellatyphimurium

Diagnosis of melanoma according to the methods of the present inventioncan be performed using, for example, Salmonella typhimurium as follows:A portion of a biopsied specimen suspected of melanoma is minced withscissors in tris-buffered saline (TBS) and then incubated inCa⁺⁺/Mg⁺⁺⁻-free saline containing trypsin, collagenase, and EDTA (SigmaChemicals) for 60 minutes at 37° C. to dissociate the tissue intoindividual cells. The cells are then rinsed free of the dissociationenzymes by centrifugation. The cells are resuspended in 1 ml DMEM/10%FBS and added to a 24 well Corning tissue culture chamber containingcover slips in the wells. The cells are then incubated in a gassed (5%CO₂/95% air) humidified incubator for 3 hours at 37° C. to allow forattachment to the cover slip.

After attachment of the biopsied cells is achieved, an attenuated,super-infective, melanoma-specific strain of Salmonella typhimurium(10⁶-10⁷ c. f.u./ml) is added. The bacteria and biopsied cells areincubated together at 37° C. for 15 minutes to allow for infection ofmelanoma cells by the S. typhimurium, and the cells are then rinsed withTBS to remove non-infecting bacteria. The cells are then permeablizedwith 0.01% saponin in 3% bovine serum albumin for 5 min, stained for DNAfor 10 minutes with 2.5 mg/ml 4′-6 Diamidino-2-phenyhndole (DAPI) andsaponin (0.01%) in TBS, washed with TBS, mounted in MOWIO™ polymermounting agent (Calbiochem) containing 1, 4-Diazabicyclo (2,2,2) octane(DABCO, Kodak) and observed by phase and fluorescence microscopy. Thepresence of DAPI-stain in the cytoplasm of the biopsied cells wouldindicate that they were melanoma cells, i.e., cells that were infectedby the melanoma-specific S. typhimurium are melanoma cells rather thanmelanocytes.

23. EXAMPLE Melanoma Tumor Targeting By Listeria Monocytogenes

This example demonstrates that Listeria monocytogenes targets to andproliferates in tumor cells when administered to melanoma bearinganimals.

C57B/6 mice were inoculated s.c. in the left flank with 5×10⁵ B16F10melanoma cells. When the tumors reached about 1-2 g (16 days postimplantation of tumor cells) the animals were inoculated i.p. with 7×10⁵cfu of Listeria monocytogenes wild type strain 43251. Prior toinoculation into mice, the Listeria culture was grown overnight in LBmedia to an OD₆₀₀ of 0.25. At the times indicated animals weresacrificed and the tumors and livers were removed, homogenized andquantitated for bacterial numbers by plating serial dilutions onto L.B.plates.

Tumors were analyzed at 24, 48, and 96 hours post-inoculation ofListeria monocytogenes. Results are shown in Table 20(I).

TABLE 20(I) AMPLIFICATION OF LISTERIA MONOCYTOGENES IN C57B/6MICE-BEARING B16F10 MELANOMA TUMORS Time L. monocytogenes/g tissue:Post-inoc. Tumor Liver Tumor:Liver 24 hrs 1.5 ± 1.4 × 10³ 8.0 ± 6.1 ×10⁴ 1:5 48 hrs 6.3 ± 8.5 × 10² 1.3 ± 1.0 × 10⁵  1:210 96 hrs 5.2 ± 6.8 ×10⁵ 5.7 ± 9.0 × 10⁵ 1:1 The results represent the average ± SD oftriplicate determinations.

As shown in Table 20(I) it was found that the levels of bacteria withinthe tumors rose about 100 fold during this time period, indicating thatwild type Listeria monocytogenes can target tumors and proliferatewithin them. Listeria monocytogenes strain 43251 was virulent in theC57B/6 mice, causing death about 5 days pot-inoculation i.p. of 7×10⁵cfu.

24. EXAMPLE Leishmania Amazonensis Shows Tumor Cell Specificity

24.1. Leishmania Amazonensis Specifically Attaches to Human MelanomaCells in Vitro

Leishmania amazonensis trypomastigotes are regarded as being highlybiospecific, in that they are unable to infect virtually any cell typesother than macrophages. Since human melanomas are known to express somemacrophage-like traits it was determined whether Leishmania amazonensiswould be able to enter into human melanoma cells in culture. Leishmaniaamazonensis promastigotes were grown in Schneider's Drosophila media(GIBCO BRL) containing 15% heat-inactivated fetal calf serum at 24° C.until the parasites were in late log phase (usually 3 to 4 days). Animalcells used in the L. amazonensis infection assays were a mouse melanomacell line which forms non-metastatic tumors when injected into C57B6mice (B16/F1), two human metastatic melanoma cell lines (M2 and M2-A7,and as a negative control human foreskin fibroblasts, HFF. Thesedifferent cell types were grown on glass coverslips in 24 well plates oron plastic Lab-Tec® (Nunc) slides in MEM culture medium with 10% fetalcalf serum, for HFF cells; Ham's F10 medium with 10% horse serum forB16/F1 cells; and DMEM with 10% fetal calf serum buffered with 10 mMHEPES, for M2 and M2-A7 cells.

The Leishmania parasites were pre-incubated for about one hour with 5%normal human serum and the cultured cells were infected with 0.5 to5.0×10⁶ parasites/ml for about two hours at 32° C. After incubation thecells were washed twice with phosphate buffered saline (PBS) and fixedwith 3% paraformaldehyde for about 30 minutes at about 4° C. Ananti-Leishmanial antibody was incubated with the fixed cells at a normalworking dilution (1:100,000) in PBS with 3% bovine serum albumen (BSA)for about one hour. After washing, a fluorescent-conjugated anti-mouseantibody (Boehringer Mannheim) was incubated with the cells at a normalworking dilution (1:500) for about one hour and then washed from thecells. The cells were then permeabilized with 0.02% Saponin (Sigma, adetergent used to remove lipids, thereby allowing penetration byantibodies) in Tris buffered saline (TBS) for 10min and stained for DNAwith 5.0 mg/ml DAPI stain (Sigma) in TBS with 0.02% Saponin. The cellswere washed with TBS and mounted on glass slides using MOWIOL™ polymermounting agent (CalBiochem) with DABCO (Kodak, a compound that sustainsfluorescent emissions). The presence of internalized parasites wasdetermined by failure to react with an anti-Leishmania monoclonalantibody in the absence of host cell membrane permeabilization.

Observations made immediately after addition of the live parasites usingan inverted phase microscope showed that among these cell lines, motileparasites were immediately adherent only when they encountered themetastatic melanoma cells, suggesting that the metastatic cells maypossess an appropriate receptor for Leishmania. These results are shownin FIG. 13.

To determine whether or not the Leishmania parasites were internalized,M2 human melanoma cells were grown, infected with Leishmania and fixedas described above with 3% paraformaldehyde at 4° C. for about 30minutes without permeabilization, washed and immunostained with amonoclonal antibody directed toward a Leishmania surface protein,followed by a rhodamine-conjugated anti-mouse antibody. After washing inTris-buffered saline (TBS), the cells were permeablized with 0.01%saponin in 3% BSA for 5 minutes, and stained for DNA 10 minutes with 2.5mg/ml 4′-6 Diamidino-2-phenylindole (DAPI) and 0.01% saponin in TBS,washed with TBS, mounted in MOWIOL™ polymer mounting agent (CaIbiochem)containing 1, 4-Diazabicyclo (2,2,2) octane (DABCO, Kodak) and observedby phase and fluorescence microscopy. This procedure detects allparasites and distinguishes between those which are internalized(inaccessible to antibody staining in non-permeabilized cells), andthose which are attached but not internalized.

Parasites were internalized by M2 cells (data not shown).Internalization was estimated to occur in 3% of the melanoma cells.These findings demonstrate that a) live Leishmania parasites were ableto enter the melanoma cells, and b) possibly only a sub-population ofthe melanoma cells were involved in the process.

24.2. Lysosomal Fusion Follows Internalization of Leishmania By Melanoma

In the normal course of invasion of macrophages by Leishmania, lysosomesfuse with the phagosome. To determine whether or not this also occurswhen Leishmania invade melanoma cells, parasites were co-localized witha lysosomal glycoprotein (lgp) marker. The cells were grown, infectedand fixed as described above except the cells were immunostained with amonoclonal antibody directed against a human lysosomal glycoprotein,LAMP-1, followed by a rhodamine-conjugated antimouse antibody. Afterwashing in TBS, the cells were stained for DNA 10 minutes with 2.5 mg/mlDAPI, washed with TBS, mounted in MOWIOL™ polymer mounting agentcontaining DABCO and observed by phase and fluorescence microscopy.Parasites co-localizing with LAMP-1 are shown in FIGS. 14A-C.Co-localization corroborates the internalization of the parasite anddemonstrates that the process of lysosomal fusion occurs when Leishmaniais internalized into the melanoma cells. In summary, Leishmaniaamazonensis in its wild type state shows invasion ability for humanmelanoma cells that has been heretofore unreported.

25. EXAMPLE Diagnosis of Melanoma In Human Tissue Biopsies UsingLeishmania Amazonensis

Diagnosis of melanoma according to the methods of the present inventioncan be performed using, for example, Leishmaina Amazonensis as follows:A portion of a biopsied specimen suspected of melanoma is minced withscissors in tris-buffered saline (TBS) and then incubated inCa⁺⁺/Mg⁺⁺-free saline containing trypsin, collagenase, and EDTA at 37°C. for 60 minutes to dissociate the tissue into individual cells. Thecells are then rinsed free of the dissociation enzymes bycentrifugation. The cells are resuspended in 1 ml DMEM/10% FBS and addedto a 24 well Corning tissue culture chamber containing cover slips inthe wells. The cells are then incubated in a gassed, 5% CO₂, humidifiedincubator at 37° C. for about three hours to allow for attachment to thecover slip.

After attachment of the biopsied cells is achieved, a melanoma-specificstrain of Leishmania amazonensis promastigotes which has been isolatedaccording to the methods of the present invention is added. Theparasites and biopsied cells are incubated together at 37° C. for abouttwo hours to allow for infection of melanoma cells by the Leishmaniaamazonensis and the cells are then rinsed with TBS to removenon-infecting parasites. The cells are then permeablized with 0.01%saponin in 3% bovine serum albumin for five minutes, stained for DNAwith 2.5 mg/ml 4′-6 Diamiclino-2-phenylindole (DAPI) and saponin (0.01%)in TBS for 10 minutes, washed with TBS, mounted in MOWIOL™ polymermounting agent (Calbiochem) containing 1,4-Diazabicyclo (2,2,2) octane(DABCO, Kodak) and observed by phase and fluorescence microscopy. Thepresence of DAPI-stain in the cytoplasm of the biopsied cells wouldindicate that they were melanoma cells.

26. EXAMPLE Diagnosis of Human Tissue Biopsies For Melanoma UsingMycobacterium Avium

Mycobacterium avium were found associated with the melanoma cells butnot with the normal melanocytes. This discriminatory ability formelanoma cells demonstrates the ability of Mycobacterium avium as avector in the diagnosis and treatment of melanoma. Diagnosis of melanomausing Mycobacterium avium is as follows: A portion of a biopsiedspecimen suspected of melanoma is minced with scissors in tris-bufferedsaline (TBS) and then incubated in Ca⁺⁺/Mg⁺⁺-free saline containingtrypsin, collagenase, and EDTA at 37° C. for 60 minutes to dissociatethe tissue into individual cells. The cells are then rinsed free of thedissociation enzymes by centrifugation. The cells are resuspended in 1ml DMEM/10% FBS and plated onto 12 mm glass cover slips in 24 wellplates with 1×10⁵ cells per well. The cells are then incubated in agassed 5% CO₂, humidified incubator 37° C. for 3 hours to allow forattachment of the cells to the cover slip.

After attachment of the biopsied cells is achieved, a melanoma-specificstrain of Mycobacterium avium which has been isolated by the methods ofthe present invention is added. The bacteria and biopsied cells areincubated together at 37° C. for 15 minutes for infection of melanomacells by the Mycobacterium avium. The cells are then rinsed with TBS toremove non-infecting bacteria. The cells are then permeablized with0.01% saponin in 3% bovine serum albumin for five minutes, stained forDNA with 2.5 mg/ml 4′-6 Diamidino-2-phenylindole (DAPI) and saponin(0.01%) in TBS for 10 minutes, washed with TBS, mounted in MOWIOL™polymer mounting agent (Calbiochem) containing 1, 4-Diazabicyclo (2,2,2)octane (DABCO, Kodak) and observed by phase and fluorescence microscopy.The presence of DAPI-stain in the cytoplasm of the biopsied cells wouldindicate that they were melanoma cells.

27. DEPOSIT OF MICROORGANISMS

The following microorganisms were deposited with the American TypeCulture Collection (ATCC), Manassas, Va. on Jun. 1, 1995 and have beenassigned the indicated Accession numbers:

Microorganism ATCC Accession No. Clone #70 55686 Clone #71 55685 Clone#72 55680 Clone #72⁵⁻³⁻² 97179 Population #72^(pop-1) 55684 Population#72^(pop-2) 55683 Population #14028^(pop-1) 55681 Population#14028^(pop-2) 55682

The following microorganisms were deposited with the American TypeCulture Collection (ATCC), Manassas, Va. on May 30, 1996, and have beenassigned the indicated Accession numbers:

Microorganism ATCC Accession No. Clone YS721 55788 Clone YS7211 55787Clone YS7212 55789 Clone YS7213 55786

The following plasmids were deposited with the American Type CultureCollection (ATCC), Manassas, Va. on May 30, 1996, and have been assignedthe indicated Accession numbers:

Microorganism ATCC Accession No. pTK-Sec3 97592 pCD-Sec1 97593pSP-SAD4-5 97591

The invention claimed and described herein is not to be limited in scopeby the specific embodiments herein disclosed since these embodiments areintended as illustrations of several aspects of the invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

A number of references are cited herein, the entire disclosures of whichare incorporated herein, in their entirety, by reference.

10 27 base pairs nucleic acid single linear DNA 1 GATCATGCAT GGCTTCGTACCCCGGCC 27 28 base pairs nucleic acid single linear DNA 2 CTAGATGCATCAGTGGCTAT GGCAGGGC 28 31 base pairs nucleic acid single linear DNA 3CTAGACTAGT TTGTCAATAA TGACAACACC C 31 30 base pairs nucleic acid singlelinear DNA 4 GATCGGATCC TTGCCCGGCG CGGCGGCCTG 30 32 base pairs nucleicacid single linear DNA 5 CTAGAAGCTT ATAAGGGTTG ATCTTTGTTG TC 32 31 basepairs nucleic acid single linear DNA 6 GTACGATATC CAGAACGATG TGCATAGCCTG 31 9 amino acids amino acid unknown peptide 7 Tyr Thr Ser Gly Tyr AlaHis Arg Ser 1 5 6 amino acids amino acid unknown peptide 8 Ser Gly TyrArg Ile Pro 1 5 25 base pairs nucleic acid single linear DNA 9GATCATGCAT GTGGAGGCTA ACAGT 25 30 base pairs nucleic acid single linearDNA 10 CTAGATGCAT CAGACAGCCG CTGCGAAGGC 30

What is claimed is:
 1. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of tumor specific microorganisms, which replicate preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the tumor specific microorganisms grow under both aerobic and anaerobic conditions, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Shigella species, wherein the tumor specific microorganisms are super-infective, tumor specific microorganisms.
 2. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of tumor specific microorganisms, which replicate preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the specific microorganisms grow under both aerobic and anaerobic conditions, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Shigella species, wherein the tumor specific microorganisms are attenuated.
 3. The method according to claim 1 wherein the tumor specific microorganisms are auxotrophic mutants.
 4. The method according to claim 2 wherein the attenuated microorganisms express an altered lipid A molecule.
 5. The method according to claim 2 wherein the attenuated microorganisms induce TNF-α expression in monocytes or macrophages from about 1 to about 75 percent compared to non-attenuated microorganisms.
 6. The method according to claim 2 wherein the tumor specific microorganisms are auxotrophic mutants.
 7. The method according to claim 1 wherein the super-infective, tumor specific microorganisms are produced by: (a) exposing a cell culture of a solid tumor cancer to a microorganism for a time sufficient so that the microorganism can infect the tumor cells; (b) allowing the microorganism to replicate, thereby producing super-infective, tumor specific microorganisms; and (c) isolating the super-infective, tumor specific microorganisms from the infected cell culture.
 8. The method according to claim 1 wherein the tumor specific microorganisms are produced by: (a) exposing a microorganism to tumor cell conditioned medium for a time sufficient to allow the microorganism to chemotact towards the tumor cell conditioned medium; (b) allowing the microorganism to replicate, thereby producing microorganisms which chemotact towards the tumor cell conditioned medium; and (c) isolating the microorganisms which chemotact towards the tumor cell conditioned medium.
 9. The method according to claim 1 wherein the super-infective, tumor specific microorganisms are produced by: (a) exposing a mammal having a solid tumor cell cancer to a microorganism for a time sufficient so that the microorganism can infect the tumor cells; (b) allowing the microorganism to replicate, thereby producing super-infective, tumor specific microorganisms; and (c) isolating the super-infective, tumor specific microorganisms from the infected tumor cells.
 10. The method according to claim 2 wherein the tumor specific microorganisms are produced by: (a) exposing a microorganism to tumor cell conditioned medium for a time sufficient to allow the microorganism to chemotact towards the tumor cell conditioned medium; (b) allowing the microorganism to replicate, thereby producing microorganisms which chemotact towards the tumor cell conditioned medium; and (c) isolating the microorganisms which chemotact towards the tumor cell conditioned medium.
 11. The method according to claim 1 or 2 wherein the effective amount is from about 1 to 1×10⁸ c.f.u./kg.
 12. The method according to claim 1 or 2 wherein the effective amount is from about 1 to 1×10² c.f.u./kg.
 13. The method according to claim 1 or 2 wherein the tumor specific microorganisms are genetically engineered.
 14. The method according to claim 7, 8 or 9 or 10 further comprising: subjecting the microorganism to mutagenesis before step (a).
 15. The method according to claim 7, 8 or 9 or 10 further comprising: (c) repeating steps (a) and (b) a desired number of times.
 16. The method according to claim 1, 2, 7, 8, 9 or 10 wherein the solid tumor cancer is melanoma cancer.
 17. The method according to claim 1, 2, 7, 8, 9 or 10 wherein the solid tumor cancer is colon carcinoma cancer.
 18. The method according to claim 1, 2, 7, 8, 9 or 10 wherein the solid tumor cancer is selected from the group consisting of lung cancer, liver cancer, kidney cancer, prostate cancer and breast cancer.
 19. The method according to claims 1, 2, 7, 8, 9, or 10 wherein the solid tumor cancer is a metastatic cancer.
 20. The method according to claim 13 wherein the tumor specific microorganisms express a suicide gene.
 21. The method according to claim 20 wherein the tumor specific microorganisms express a suicide gene selected from the group consisting of p450 oxidoreductase, HSV thymidine kinase, E. coli cytosine deaminase, carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase and carboxypeptidase A.
 22. The method according to claim 20 wherein expression of the suicide gene is controlled by a constitutive promoter, an inducible promoter, or a tumor cell specific promoter.
 23. The method according to claim 20 wherein the suicide gene is encoded by the open reading frame of the insert of a plasmid selected from the group consisting of pTK-Sec3, pCD-Sec1 and pSP-SAD4-5.
 24. The method according to claim 23 wherein the suicide gene is selected from the group consisting of HSV thymidine kinase and E. coli cytosine deaminase.
 25. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms grows under both aerobic and anaerobic conditions, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Shigella species, wherein the single colony clone of an isolated population of tumor specific microorganisms is a single colony clone of an isolated population of super-infective, tumor specific microorganisms.
 26. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms grows under both aerobic and anaerobic conditions, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Shigella species, wherein the tumor specific single colony clone is attenuated.
 27. The method according to claim 25 wherein the single colony clone of an isolated population of super-infective, tumor specific microorganisms is produced by: (a) exposing a cell culture of a solid tumor cancer to a microorganism for a time sufficient so that the microoranism can infect the tumor cells; (b) isolating a population of super-infective, tumor specific microorganisms from the infected cell culture; and (c) culturing the population isolated in step (b) so that single colony clones are obtained.
 28. The method according to claim 25 wherein the single colony clone of an isolated population of tumor specific microorganisms is produced by: (a) exposing a microorganism to tumor cell conditioned medium for a time sufficient to allow the microorganism to chemotact towards the tumor cell conditioned medium; (b) isolating a population of microorganisms which chemotacts towards the tumor cell conditioned medium; and (c) culturing the population isolated in step (b) so that single colony clones are obtained.
 29. The method according to claim 25 wherein the single colony clone of an isolated population of super-infective, tumor specific microorganisms is produced by: (a) exposing a mammal having a solid tumor cell cancer to a microorganism for a time sufficient so that the microorganism can infect the tumor cells; (b) isolating a population of super-infective, tumor specific microorganisms from the infected tumor cells; and (c) culturing the population isolated in step (b) so that single colony clones are obtained.
 30. The method according to claim 25 or 26 wherein the single colony clone is genetically engineered.
 31. The method according to claim 25 or 26 wherein the effective amount is from about 1 to 1×10⁸ c.f.u./kg.
 32. The method according to claim 25 or 26 wherein the effective amount is from about 1 to 1×10² c.f.u./kg.
 33. The method according to claim 26 wherein the single colony clone of an isolated population of tumor specific microorganisms is produced by: (a) exposing a microorganism to tumor cell conditioned medium for a time sufficient to allow the microorganism to chemotact towards the tumor cell conditioned medium; (b) isolating a population of microorganisms which chemotacts towards the tumor cell conditioned medium; and (c) culturing the population isolated in step (b) so that single colony clones are obtained.
 34. The method according to claim 26 wherein the attenuated clone expresses an altered lipid A molecule.
 35. The method according to claim 26 wherein the single colony clone induces TNF-α expression in monocytes or macrophages from about 1 to about 75 percent compared to a single colony clone that is not attenuated.
 36. The method according to claim 27, 28, 29 or 33 further comprising: subjecting the microorganism to mutagenesis before step (a).
 37. The method according to claim 27, 28, 29 or 33 further comprising: (d) repeating steps (a) and (b) a desired number of times before step (c).
 38. The method according to claim 25, 26, 27, 28, 29 or 33 wherein the solid tumor cancer is melanoma cancer.
 39. The method according to claim 25, 26, 27, 28, 29 or 33 wherein the solid tumor cancer is colon carcinoma cancer.
 40. The method according to claim 25, 26, 27, 28, 29 or 33 wherein the solid tumor cancer is selected from the group consisting of lung cancer, liver cancer, kidney cancer, prostate cancer and breast cancer.
 41. The method according to claims 25, 26, 27, 28, 29, or 33 wherein the solid tumor cancer is a metastatic cancer.
 42. The method according to claim 30 wherein the single colony clone expresses a suicide gene.
 43. The method according to claim 42 wherein expression of the suicide gene is controlled by a constitutive promoter, an inducible promoter, or a tumor cell specific promoter.
 44. The method according to claim 42 wherein the suicide gene is encoded by the open reading frame of the insert of a plasmid selected from the group consisting of pTK-Sec3, pCD-Sec1 and pSP-SAD4-5.
 45. The method according to claim 44 wherein the suicide gene is selected from the group consisting of HSV thymidine kinase and E. coli cytosine deaminase.
 46. The method according to claim 42 wherein the single colony clone expresses a suicide gene selected from the group consisting of p450 oxidoreductase, HSV thymidine kinase, E. coli cytosine deaminase, carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase and carboxypeptidase A.
 47. The method according to claim 46 wherein the suicide gene is selected from the group consisting of HSV thymidine kinase and E. coli cytosine deaminase.
 48. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms grows under both aerobic and anaerobic conditions, and wherein the single colony clone expresses a suicide gene which gene is controlled by an inducible promoter, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Shigella species.
 49. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms, which grows under both aerobic and anaerobic conditions, and wherein the single colony clone expresses a suicide gene which gene is controlled by a bacterial promoter that is activated in specific tumor cells, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Shigella species.
 50. A diagnostic kit comprising an effective amount of a tumor-specific microorganism and instructions for use for in vitro diagnosis of a solid tumor cancer, wherein the tumor specific microorganism is a tumor specific Shigella species.
 51. The diagnostic kit according to claim 50 further comprising non-cancerous counterpart control cells.
 52. The diagnostic kit according to claim 50 wherein the tumor-specific microorganism is a super-infective, tumor-specific microorganism.
 53. The diagnostic kit according to claim 52 wherein the microorganism is genetically engineered.
 54. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of an tumor specific microorganisms, which replicate preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the tumor specific microorganisms grow under both aerobic and anaerobic conditions, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Streptococcus species genetically engineered to express a suicide gene.
 55. The method according to claim 54 wherein the effective amount is from about 1 to 1×10⁸ c.f.u./kg.
 56. The method according to claim 54 wherein the effective amount is from about 1 to 1×10² c.f.u./kg.
 57. The method according to claim 54 wherein the tumor specific microorganisms are auxotrophic mutants.
 58. The method according to claim 54 wherein the tumor specific microorganisms are super-infective, tumor specific microorganisms.
 59. The method according to claim 54 wherein the tumor specific microorganisms are attenuated.
 60. The method according to claim 54 wherein the tumor specific microorganisms are produced by: (a) exposing a microorganism to tumor cell conditioned medium for a time sufficient to allow the microorganism to chemotact towards the tumor cell conditioned medium; (b) allowing the microorganism to replicate, thereby producing microorganisms which chemotact towards the tumor cell conditioned medium; and (c) isolating the microorganisms which chemotact towards the tumor cell conditioned medium.
 61. The method according to claims 54, 63, 60 or 64 wherein the solid tumor cancer is a metastatic cancer.
 62. The method according to claim 59 wherein the attenuated microorganisms induce TNF-α expression in monocytes or macrophages from about 1 to about 75 percent compared to non-attenuated microorganisms.
 63. The method according to claim 58 wherein the super-infective, tumor specific microorganisms are produced by: (a) exposing a cell culture of a solid tumor cancer to a microorganism for a time sufficient so that the microorganism can infect the tumor cells; (b) allowing the microorganism to replicate, thereby producing super-infective, tumor specific microorganisms; and (c) isolating the super-infective, tumor specific microorganisms from the infected cell culture.
 64. The method according to claim 58 wherein the super-infective, tumor specific microorganisms are produced by: (a) exposing a mammal having a solid tumor cell cancer to a microorganism for a time sufficient so that the microorganism can infect the tumor cells; (b) allowing the microorganism to replicate, thereby producing super-infective, tumor specific microorganisms; and (c) isolating the super-infective, tumor specific microorganisms from the infected tumor cells.
 65. The method according to claim 59 wherein the attenuated population expresses an altered lipid A molecule.
 66. The method according to claim 58 wherein the tumor specific microorganisms are auxotrophic mutants.
 67. The method according to claim 63, 60 or 64 further comprising: subjecting the microorganism to mutagenesis before step (a).
 68. The method according to claim 63, 60 or 64 further comprising: (c) repeating steps (a) and (b) a desired number of times.
 69. The method according to claim 54, 63, 60 or 64 wherein the solid tumor cancer is melanoma cancer.
 70. The method according to claim 54, 63, 60 or 64 wherein the solid tumor cancer is colon carcinoma cancer.
 71. The method according to claim 54, 63, 60 or 64 wherein the solid tumor cancer is selected from the group consisting of lung cancer, liver cancer, kidney cancer, prostate cancer and breast cancer.
 72. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms grows under both aerobic and anaerobic conditions, and wherein the single colony clone expresses a suicide gene which gene is controlled by an inducible promoter, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Streptococcus species genetically engineered to express a suicide gene.
 73. The method according to claim 72 wherein the effective amount is from about 1 to 1×10⁸ c.f.u./kg.
 74. The method according to claim 72 wherein the effective amount is from about 1 to 1×10² c.f.u./kg.
 75. The method according to claim 72 wherein the single colony clone of an isolated population of tumor specific microorganisms is a single colony clone of an isolated population of super-infective, tumor specific microorganisms.
 76. The method according to claim 72 wherein the tumor specific single colony clone is attenuated.
 77. The method according to claim 72 wherein the single colony clone of an isolated population of tumor specific microorganisms is produced by: (a) exposing a microorganism to tumor cell conditioned medium for a time sufficient to allow the microorganism to chemotact towards the tumor cell conditioned medium; (b) isolating a population of microorganisms which chemotacts towards the tumor cell conditioned medium; and (c) culturing the population isolated in step (b) so that single colony clones are obtained.
 78. The method according to claim 72 or 75 wherein the suicide gene is encoded by the open reading frame of the insert of a plasmid selected from the group consisting of pTK-Sec3, pCD-Sec1 and pSP-SAD4-5.
 79. The method according to claim 72 or 75 wherein the suicide gene selected from the group consisting of p450 oxidoreductase, HSV thymidine kinase, E. coli cytosine deaminase, carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase and carboxypeptidase A.
 80. The method according to claim 72 or 75 wherein expression of the suicide gene is controlled by a constitutive promoter, an inducible promoter, or a tumor cell specific promoter.
 81. The method according to claim 78 wherein the suicide gene is selected from the group consisting of HSV thymidine kinase and E. coli cytosine deaminase.
 82. The method according to claim 79 wherein the suicide gene is selected from the group consisting of HSV thymidine kinase and E. coli cytosine deaminase.
 83. The method according to claim 76 wherein the single colony clone induces TNF-α expression in monocytes or macrophages from about 1 to about 75 percent compared to a single colony clone that is not attenuated.
 84. The method according to claim 75 wherein the single colony clone of an isolated population of super-infective, tumor specific microorganisms is produced by: (a) exposing a cell culture of a solid tumor cancer to a microorganism for a time sufficient so that the microorganism can infect the tumor cells; (b) isolating a population of super-infective, tumor specific microorganisms from the infected cell culture; and (c) culturing the population isolated in step (b) so that single colony clones are obtained.
 85. The method according to claim 75 wherein the single colony clone of an isolated population of super-infective, tumor specific microorganisms is produced by: (a) exposing a mammal having a solid tumor cell cancer to a microorganism for a time sufficient so that the microorganism can infect the tumor cells; (b) isolating a population of super-infective, tumor specific microorganisms from the infected tumor cells; and (c) culturing the population isolated in step (b) so that single colony clones are obtained.
 86. The method according to claim 84, 77 or 85 further comprising: subjecting the microorganism to mutagenesis before step (a).
 87. The method according to claim 84, 77 or 85 further comprising: (d) repeating steps (a) and (b) a desired number of times before step (c).
 88. The method according to claim 72, 84, 77 or 85 wherein the solid tumor cancer is melanoma cancer.
 89. The method according to claim 72, 84, 77 or 85 wherein the solid tumor cancer is colon carcinoma cancer.
 90. The method according to claim 72, 84, 77 or 85 wherein the solid tumor cancer is selected from the group consisting of lung cancer, liver cancer, kidney cancer, prostate cancer and breast cancer.
 91. The method according to claims 72, 84, 77 or 85 wherein the solid tumor cancer is a metastatic cancer.
 92. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms that grows under both aerobic and anaerobic conditions, and wherein the single colony clone expresses a suicide gene which gene is controlled by an inducible promoter, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Streptococcus species genetically engineered to express a suicide gene.
 93. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms, which grows under both aerobic and anaerobic conditions, and wherein the single colony clone expresses a suicide gene which gene is controlled by a bacterial promoter that is activated in specific tumor cells, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Streptococcus species genetically engineered to express a suicide gene.
 94. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of tumor specific microorganisms, which replicate preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the tumor specific microorganisms grow under both aerobic and anaerobic conditions, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, wherein the tumor specific microorganisms are a tumor specific Streptococcus species, and wherein the administration is oral, topical, intravenous, intraperitoneal, subcutaneous, or intramuscular.
 95. The method according to claim 94 wherein the effective amount is from about 1 to 1×10⁸ c.f.u./kg.
 96. The method according to claim 94 wherein the effective amount is from about 1 to 1×10² c.f.u./kg.
 97. The method according to claim 94 wherein the tumor specific microorganisms are auxotrophic mutants.
 98. The method according to claim 94 wherein the tumor specific microorganisms are super-infective, tumor specific microorganisms.
 99. The method according to claim 94 wherein the tumor specific microorganisms are attenuated.
 100. The method according to claim 94 wherein the tumor specific microorganisms are produced by: (a) exposing a microorganism to tumor cell conditioned medium for a time sufficient to allow the microorganism to chemotact towards the tumor cell conditioned medium; (b) allowing the microorganism to replicate, thereby producing microorganisms which chemotact towards the tumor cell conditioned medium; and (c) isolating the microorganisms which chemotact towards the tumor cell conditioned medium.
 101. The method according to claim 99 wherein the attenuated microorganisms induce TNF-α expression from about 1 to about 75 percent compared to non-attenuated microorganisms.
 102. The method according to claim 98 wherein the super-infective, tumor specific microorganisms are produced by: (a) exposing a cell culture of a solid tumor cancer to a microorganism for a time sufficient so that the microorganism can infect the tumor cells; (b) allowing the microorganism to replicate, thereby producing super-infective, tumor specific microorganisms; and (c) isolating the super-infective, tumor specific microorganisms from the infected cell culture.
 103. The method according to claim 98 wherein the super-infective, tumor specific microorganisms are produced by: (a) exposing a mammal having a solid tumor cell cancer to a microorganism for a time sufficient so that the microorganism can infect the tumor cells; (b) allowing the microorganism to replicate, thereby producing super-infective, tumor specific microorganisms; and (c) isolating the super-infective, tumor specific microorganisms from the infected tumor cells.
 104. The method according to claim 102, 100 or 103 further comprising: subjecting the microorganism to mutagenesis before step (a).
 105. The method according to claim 102, 100 or 103 further comprising: (c) repeating steps (a) and (b) a desired number of times.
 106. The method according to claim 94, 102, 100 or 103 wherein the solid tumor cancer is melanoma cancer.
 107. The method according to claim 94, 102, 100 or 103 wherein the solid tumor cancer is colon carcinoma cancer.
 108. The method according to claim 94, 102, 100 or 103 wherein the solid tumor cancer is selected from the group consisting of lung cancer, liver cancer, kidney cancer, prostate cancer and breast cancer.
 109. The method according to claims 94, 102, 100 or 103 wherein the solid tumor cancer is a metastatic cancer.
 110. The method according to claim 98 wherein the tumor specific microorganisms are auxotrophic mutants.
 111. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms grows under both aerobic and anaerobic conditions, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, wherein the tumor specific microorganisms are a tumor specific Streptococcus species, and wherein the administration is oral, topical, intravenous, intraperitoneal, subcutaneous, or intramuscular.
 112. The method according to claim 111 wherein the effective amount is from about 1 to 1×10⁸ c.f.u./kg.
 113. The method according to claim 111 wherein the effective amount is from about 1 to 1×10² c.f.u./kg.
 114. The method according to claim 111 wherein the single colony clone of an isolated population of tumor specific microorganisms is a single colony clone of an isolated population of super-infective, tumor specific microorganisms.
 115. The method according to claim 111 wherein the tumor specific single colony clone is attenuated.
 116. The method according to claim 111 or 114 wherein the single colony clone is genetically engineered.
 117. The method according to claim 111 wherein the single colony clone of an isolated population of tumor specific microorganisms is produced by: (a) exposing a microorganism to tumor cell conditioned medium for a time sufficient to allow the microorganism to chemotact towards the tumor cell conditioned medium; (b) isolating a population of microorganisms which chemotacts towards the tumor cell conditioned medium; and (c) culturing the population isolated in step (b) so that single colony clones are obtained.
 118. The method according to claim 115 wherein the single colony clone induces TNF-α expression from about 1 to about 75 percent compared to a single colony clone that is not attenuated.
 119. The method according to claim 114 wherein the single colony clone of an isolated population of super-infective, tumor specific microorganisms is produced by: (a) exposing a cell culture of a solid tumor cancer to a microorganism for a time sufficient so that the microorganism can infect the tumor cells; (b) isolating a population of super-infective, tumor specific microorganisms from the infected cell culture; and (c) culturing the population isolated in step (b) so that single colony clones are obtained.
 120. The method according to claim 114 wherein the single colony clone of an isolated population of super-infective, tumor specific microorganisms is produced by: (a) exposing a mammal having a solid tumor cell cancer to a microorganism for a time sufficient so that the microorganism can infect the tumor cells; (b) isolating a population of super-infective, tumor specific microorganisms from the infected tumor cells; and (c) culturing the population isolated in step (b) so that single colony clones are obtained.
 121. The method according to claim 119, 117 or 120 further comprising: subjecting the microorganism to mutagenesis before step (a).
 122. The method according to claim 119, 117 or 120 further comprising: (d) repeating steps (a) and (b) a desired number of times before step (c).
 123. The method according to claim 111, 119, 117 or 120 wherein the solid tumor cancer is melanoma cancer.
 124. The method according to claim 111, 119, 117 or 120 wherein the solid tumor cancer is colon carcinoma cancer.
 125. The method according to claim 111, 119, 117 or 120 wherein the solid tumor cancer is selected from the group consisting of lung cancer, liver cancer, kidney cancer, prostate cancer and breast cancer.
 126. The method according to claim 111, 119, 117 or 120 wherein the solid tumor cancer is a metastatic cancer.
 127. The method according to claim 116 wherein the single colony clone expresses a suicide gene.
 128. The method according to claim 127 wherein expression of the suicide gene is controlled by a constitutive promoter, an inducible promoter, or a tumor cell specific promoter.
 129. The method according to claim 127 wherein the suicide gene is encoded by the open reading frame of the insert of a plasmid selected from the group consisting of pTK-Sec3, pCD-Sec1 and pSP-SAD4-5.
 130. The method according to claim 127 wherein the single colony clone expresses a suicide gene selected from the group consisting of p450 oxidoreductase, HSV thymidine kinase, E. coli cytosine deaminase, carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase and carboxypeptidase A.
 131. The method according to claim 129 wherein the suicide gene is selected from the group consisting of HSV thymidine kinase and E. coli cytosine deaminase.
 132. The method according to claim 130, wherein the suicide gene is selected from the group consisting of HSV thymidine kinase and E. coli cytosine deaminase.
 133. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms grows under both aerobic and anaerobic conditions, and wherein the single colony clone expresses a suicide gene which gene is controlled by an inducible promoter, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, wherein the tumor specific microorganisms are a tumor specific Streptococcus species, and wherein the administration is oral, topical, intravenous, intraperitoneal, subcutaneous, or intramuscular.
 134. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms, which grows under both aerobic and anaerobic conditions, and wherein the single colony clone expresses a suicide gene which gene is controlled by a bacterial promoter that is activated in specific tumor cells, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, wherein the tumor specific microorganisms are a tumor specific Streptococcus species, and wherein the administration is oral, topical, intravenous, intraperitoneal, subcutaneous, or intramuscular.
 135. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the tumor specific microorganisms grow under both aerobic and anaerobic conditions, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Listeria monocytogenes, wherein the tumor specific microorganisms are super-infective, tumor specific microorganisms.
 136. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of tumor specific microorganisms, which replicate preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the specific microorganisms grow under both aerobic and anaerobic conditions, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Listeria monocytogenes, wherein the tumor specific microorganisms are attenuated.
 137. The method according to claim 135 wherein the tumor specific microorganisms are auxotrophic mutants.
 138. The method according to claim 136 wherein the attenuated microorganisms induce TNF-α expression in monocytes or macrophages from about 1 to about 75 percent compared to non-attenuated microorganisms.
 139. The method according to claim 135 wherein the super-infective, tumor specific microorganisms are produced by: (a) exposing a cell culture of a solid tumor cancer to a microorganism for a time sufficient so that the microorganism can infect the tumor cells; (b) allowing the microorganism to replicate, thereby producing super-infective, tumor specific microorganisms; and (c) isolating the super-infective, tumor specific microorganisms from the infected cell culture.
 140. The method according to claim 135 wherein the tumor specific microorganisms are produced by: (a) exposing a microorganism to tumor cell conditioned medium for a time sufficient to allow the microorganism to chemotact towards the tumor cell conditioned medium; (b) allowing the microorganism to replicate, thereby producing microorganisms which chemotact towards the tumor cell conditioned medium; and (c) isolating the microorganisms which chemotact towards the tumor cell conditioned medium.
 141. The method according to claim 135 wherein the super infective, tumor specific microorganisms are produced by: (a) exposing a mammal to having a solid tumor cell cancer to a microorganism for a time sufficient so that the microorganism can infect the tumor cells; (b) allowing the microorganism to replicate, thereby producing super-infective, tumor specific microorganisms; and (c) isolating the super-infective, tumor specific microorganisms from the infected tumor cells.
 142. The method according to claim 136 wherein the tumor specific microorganisms are produced by: (a) exposing a microorganism to tumor cell conditioned medium for a time sufficient to allow the microorganism to chemotact towards the tumor cell conditioned medium; (b) allowing the microorganism to replicate, thereby producing microorganisms which chemotact towards the tumor cell conditioned medium; and (c) isolating the microorganisms which chemotact towards the tumor cell conditioned medium.
 143. The method according to claim 135 or 136 wherein the tumor specific microorganisms are genetically engineered.
 144. The method according to claim 139, 140, 141 or 142 further comprising: subjecting the microorganism to mutagenesis before step (a).
 145. The method according to claim 139, 140, 141 or 142 further comprising: (c) repeating steps (a) and (b) a desired number of times.
 146. The method according to claim 135, 136, 139, 140, 141 or 142 wherein the solid tumor cancer is melanoma cancer.
 147. The method according to claim 135, 136, 139, 140, 141 or 142 wherein the solid tumor cancer is colon carcinoma cancer.
 148. The method according to claim 135, 136, 139, 140, 141 or 142 wherein the solid tumor cancer is selected from the group consisting of lung cancer, liver cancer, kidney cancer, prostate cancer and breast cancer.
 149. The method according to claims 135, 136, 139, 140, 141, or 142 wherein the solid tumor cancer is a metastatic cancer.
 150. The method according to claim 143 wherein the tumor specific microorganisms express a suicide gene.
 151. The method according to claim 150 wherein the tumor specific microorganisms express a suicide gene selected from the group consisting of p450 oxidoreductase, HSV thymidine kinase, E. coli cytosine deaminase, carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase and carboxypeptidase A.
 152. The method according to claim 150 wherein expression of the suicide gene is controlled by a constitutive promoter, an inducible promoter, or a tumor cell specific promoter.
 153. The method according to claim 150 wherein the suicide gene is encoded by the open reading frame of the insert of a plasmid selected from the group consisting of pTK-Sec3, pCD-Sec1 and pSP-SAD4-5.
 154. The method according to claim 153 wherein the suicide gene is selected from the group consisting of HSV thymidine kinase and E. coli cytosine deaminase.
 155. The method according to claim 135 or 136 wherein the effective amount is from about 1 to 1×10⁸ c.f.u./kg.
 156. The method according to claim 135 or 136 wherein the effective amount is from about 1 to 1×10² c.f.u./kg.
 157. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms grows under both aerobic and anaerobic conditions, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Listeria monocytogenes, wherein the single colony clone of an isolated population of tumor specific microorganisms is a single colony clone of an isolated population of super-infective, tumor specific microorganisms.
 158. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms grows under both aerobic and anaerobic conditions, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Listeria monocytogenes, wherein the tumor specific single colony clone is attenuated.
 159. The method according to claim 158 wherein the single colony clone induces TNF-α expression in monocytes or macrophages from about 1 to about 75 percent compared to a single colony clone that is not attenuated.
 160. The method according to claim 157 wherein the single colony clone of an isolated population of tumor specific microorganisms is produced by: (a) exposing a mammal having a solid tumor cell cancer to a microorganism for for a time sufficient so that the population of microorganism can infect the tumor cells; (b) isolating a population of super-infective, tumor specific microorganisms from the infected cell culture; and (c) culturing the population isolated in step (b) so that single colony clones are obtained.
 161. The method according to claim 157 wherein the single colony clone of an isolated population of tumor specific microorganisms is produced by: (a) exposing a microorganism to tumor cell conditioned medium for a time sufficient to allow the microorganism to chemotact towards the tumor cell conditioned medium; (b) isolating a population of microorganisms which chemotacts towards the tumor cell conditioned medium; and (c) cultured the population isolated in step (b) so that single colony clones are obtained.
 162. The method according to claim 157 wherein the single colony clone of an isolated population of super-infective, tumor specific microorganisms is produced by: (a) exposing a mammal having a solid tumor cell cancer to a microorganism for for a time sufficient so that the population of microorganisms can infect the tumor cells; (b) isolating a population of super-infective, tumor specific microorganisms from the infected tumor cells; and (c) culturing the population isolated in step (b) so that single colony clones are obtained.
 163. The method according to claim 158 wherein the single colony clone of an isolated population of tumor specific microorganisms is produced by: (a) exposing a microorganism to tumor cell conditioned medium for a time sufficient to allow the microorganism to chemotact towards the tumor cell conditioned medium; (b) isolating a population of microorganisms which chemotacts towards the tumor cell conditioned medium; and (c) culturing the population isolated in step (b) so that single colony clones are obtained.
 164. The method according to claim 157 or 158 wherein the single colony clone is genetically engineered.
 165. The method according to claim 157 or 158 wherein the effective amount is from about 1 to 1×10⁸ c.f.u./kg.
 166. The method according to claim 157 or 158 wherein the effective amount is from about 1 to 1×10² c.f.u./kg.
 167. The method according to claim 160, 161, 162 or 163 further comprising: subjecting the microorganism to mutagenesis before step (a).
 168. The method according to claim 160, 161, 162 or 163 further comprising: (d) repeating steps (a) and (b) a desired number of times before step (c).
 169. The method according to claim 157, 158, 160, 161, 162 or 163 wherein the solid tumor cancer is melanoma cancer.
 170. The method according to claim 157, 158, 160, 161, 162 or 163 wherein the solid tumor cancer is colon carcinoma cancer.
 171. The method according to claim 157, 158, 169, 161, 162 or 163 wherein the solid tumor cancer is selected from the group consisting of lung cancer, liver cancer, kidney cancer, prostate cancer and breast cancer.
 172. The method according to claims 157, 158, 160, 161, 162, or 163 wherein the solid tumor cancer is a metastatic cancer.
 173. The method according to claim 164 wherein the single colony clone expresses a suicide gene.
 174. The method according to claim 173 wherein expression of the suicide gene is controlled by a constitutive promoter, an inducible promoter, or a tumor cell specific promoter.
 175. The method according to claim 173 wherein the single colony clone expresses a suicide gene selected from the group consisting of p450 oxidoreductase, HSV thymidine kinase, E. coli cytosine deaminase, carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase and carboxypeptidase A.
 176. The method according to claim 173 wherein the suicide gene is encoded by the open reading frame of the insert of a plasmid selected from the group consisting of pTK-Sec3, pCD-Sec1 and pSP-SAD4-5.
 177. The method according to claim 176 wherein the suicide gene is selected from the group consisting of HSV thymidine kinase and E. coli cytosine deaminase.
 178. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms grows under both aerobic and anaerobic conditions, and wherein the single colony clone expresses a suicide gene which gene is controlled by an inducible promoter, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganims are a tumor specific Listeria monocytogenes.
 179. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms, which grows under both aerobic and anaerobic conditions, and wherein the single colony clone expresses a suicide gene which gene is controlled by a bacterial promoter that is activated in specified tumor cells, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Listeria monocytogenes.
 180. A diagnostic kit comprising an effective amount of a tumor-specific microorganism and instructions for use for in vitro diagnosis of a solid tumor cancer, wherein the tumor specific microorganism is a tumor specific Listeria monocytogenes.
 181. The diagnostic kit according to claim 180 wherein the tumor-specific microorganism is a super-infective, tumor-specific microorganism.
 182. The diagnostic kit according to claim 180 further comprising non-cancerous counterpart control cells.
 183. The diagnostic kit according to claim 181 wherein the microorganism is genetically engineered.
 184. The method according to claim 166 wherein the tumor specific microorganisms are auxotrophic mutants.
 185. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms grows under both aerobic and anaerobic conditions, and wherein the single colony clone expresses a suicide gene which gene is controlled by a constitutive promoter, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Shigella species.
 186. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms grows under both aerobic and anaerobic conditions, and wherein the single colony clone expresses a suicide gene which gene is controlled by a constitutive promoter, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Streptococcus species genetically engineered to express a suicid gene.
 187. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms grows under both aerobic and anaerobic conditions, and wherein the single colony clone expresses a suicide gene which gene is controlled by a constitutive promoter, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Streptococcus species, and wherein the administration is oral, topical, intravenous, intraperitoneal, subcutaneous, or intramuscular.
 188. A method for reducing volume or inhibiting growth of a solid tumor cancer, comprising: administering an amount of a single colony clone of an isolated population of tumor specific microorganisms, which replicates preferentially in the tumor after administration, to a patient having a solid tumor cancer, wherein the single colony clone of an isolated population of tumor specific microorganisms grows under both aerobic and anaerobic conditions, and wherein the single colony clone expresses a suicide gene which gene is controlled by a constitutive promoter, which amount is effective to reduce volume or inhibit growth of said solid tumor cancer, and wherein the tumor specific microorganisms are a tumor specific Listeria monocytogenes. 